Antibodies that bind to fibroblast activation protein alpha and death receptor 4

Abstract

The present invention relates to a multispecific antibody comprising a FAPα-binding region comprising at least a first heavy chain variable region and a first light chain variable region, and a DR4-binding region comprising a second heavy chain variable region and a second light chain variable region. The invention further provides pharmaceutical compositions comprising the antibody, and uses of the antibody in therapeutic and diagnostic procedures, particularly in cancer treatment.

Description

[Technical Field]

[0001] The present invention relates to antibodies that bind to fibroblast activation protein alpha (FAPα) and death receptor 4 (DR4). The invention further provides pharmaceutical compositions comprising the antibodies, and uses of the antibodies for therapeutic and diagnostic procedures, such as the treatment of cancer. [Background technology]

[0002] Therapeutic monoclonal antibodies are a promising class of immunotherapy due to their specific characteristics, including target specificity, immunomodulation, and generally low toxicity. Monoclonal antibodies are constructed from two distinct functional units: an antigen-binding fragment (Fab) that binds to the target antigen, and a constant fragment (Fc) that mediates antibody-dependent immune effector functions. The primary Fc domain-mediated mechanism of action is binding to Fc-gamma receptors (specific for IgG) on various immune cells, resulting in complement-dependent cytotoxicity (CDC) and, for example, antibody-dependent cell-mediated cytotoxicity (ADCC). Furthermore, Fab binding to the target can result in signal transduction perturbations.

[0003] Our knowledge of antibody-based therapeutic strategies has advanced dramatically in recent years, resulting in breakthroughs in target biology, mechanism of action, as well as antibody formats and development, which have led to improved clinical efficacy as well as improved target specificity (and therefore safety) of antibody-based therapies.

[0004] Apoptosis is a form of programmed cell death. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) can induce apoptosis through binding to its agonist receptor, which contains an intracellular death domain (DD). DR4, also known as tumor necrosis factor receptor superfamily member 10A (TNFRSF10A), TRAIL receptor 1 (TRAIL-R1), and CD261, is a cell surface receptor of the TNF receptor superfamily that binds TRAIL and mediates apoptosis. DR4 shares 60% homology with death receptor 5 (DR5), another known TRAIL receptor that can induce apoptosis. DR4 is a single-pass type I membrane protein with at least three extracellular cysteine-rich domains (CRDs), a transmembrane domain (TM), and a cytoplasmic DD. TRAIL binding leads to DR4 activation via receptor trimerization, resulting in clustering of DDs, followed by recruitment of the Fas-associated death domain (FADD) adaptor protein. FADD then recruits caspases-8 and -10 to form the death-inducing signaling complex (DISC). Active caspases-8 and -10 are then released into the cytosol, where they activate downstream effector caspases, such as caspase-3. Activation of effector caspases culminates in apoptotic cell death.

[0005] FAPα is a type II transmembrane (homodimeric) serine protease that is overexpressed in pathological conditions, including fibrosis, arthritis, and cancer. It is primarily expressed by activated stromal fibroblasts, such as CAFs. FAPα can also be excreted from the cell membrane to form soluble FAPα. FAPα is a member of the prolyl peptidase family and shares 70% amino acid sequence identity with the well-described dipeptidyl peptidase 4 (DPP4). While both peptidases contain dipeptidyl peptidase enzymatic activity, the endopeptidase activity is FAPα-specific and targets substrates, including denatured collagen and α-2 antiplasmin. For most cancers, elevated FAPα expression is associated with poor outcome, although the underlying biological mechanisms are not fully understood.

[0006] The limited antitumor efficacy of several first-generation DR agonist antibodies in clinical trials is likely due to their inability to induce efficient receptor clustering, which is essential for inducing apoptosis (Dubuisson and Micheau, Antibodies (Basel) 6(4), 2017). One such example is the DR4-specific agonist monoclonal antibody mapatumumab (HGS-ETR1), which has shown limited clinical activity when investigated in multiple phase 1 / 2 trials (Snajdauf et al., Front Mol Biosci 8:628332, 2021). Next-generation drugs, such as the TRAIL-R agonist Fc fusion protein eftozanermin alfa (ABBV-621), have shown promising clinical activity but also induced side effects such as hepatotoxicity (Papadopoulos et al., Cancer Chemother Pharmacol 75(5):887-895, 2015; LoRusso et al., Invest New Drugs 40(4):762-772, 2022; Di Cristofano et al., Biochem Soc Trans 51(1):57-70, 2023). Dual targeting of DRs and FAPα has been explored using RG7386, an optimized tetravalent bispecific antibody targeting FAPα and DR5 (U.S. Patent No. 9926379B2). However, clinical development for solid malignancies was discontinued in 2018. Therefore, there is a clear unmet need for the development of novel therapeutic agents with improved safety and efficacy. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent No. 9926379B2 [Non-patent literature]

[0008] [Non-Patent Document 1] Dubuisson and Micheau,Antibodies(Basel)6(4),2017 [Non-patent document 2] Snajdauf et al.,Front Mol Biosci 8:628332,2021 [Non-patent document 3] Papadopoulos et al, Cancer Chemother Pharmacol 75(5):887-895,2015 [Non-patent document 4] LoRusso et al.,Invest New Drugs 40(4):762-772,2022 [Non-patent document 5] Di Cristofano et al.,Biochem Soc Trans 51(1):57-70,2023 Summary of the Invention [Problem to be solved by the invention]

[0009] It is an object of the present invention to provide multispecific antibodies with improved safety and efficacy. [Means for solving the problem]

[0010] The multispecific antibody according to the present invention comprises a DR4-binding region and a FAPα-binding region. Thus, the proposed mechanism of action for the multispecific antibody according to the present invention is the conditional transactivation of DR4 as a result of simultaneous binding to DR4-expressing tumor cells and FAPα on CAFs in the tumor microenvironment (TME). Furthermore, it has been found that some DR4-binding antibodies bind well to DR4-expressing cells, and some FAPα-binding antibodies bind well to FAPα-expressing cells in a bivalent (monoclonal) format, but show reduced binding in a monovalent format. Therefore, another object of the present invention relates to monoclonal antibodies capable of strong binding in either a monovalent or bivalent format. This can form the basis for the generation of, for example, bispecific antibodies that show strong monovalent binding to DR4 and FAPα.

[0011] In the present invention, as demonstrated, for example, by Examples 10-13 and 15, the trans-binding mechanism of a multispecific antibody is advantageous when killing of DR4-expressing tumor cells can depend on simultaneous binding of the multispecific antibody to FAPα-expressing cells, such as CAFs. Thus, FAPα-dependent DR4 transactivation leads to improved tumor-specific targeting due to the high expression of FAPα on CAFs in the TME. Furthermore, multispecific antibodies have demonstrated an expanded therapeutic window through trans-binding-dependent DR4 activation, as opposed to cis-binding, when DR4 is not expressed on fibroblasts (e.g., Example 9). Because fibroblasts do not express DR4, they are not susceptible to induction of cell death by FAP-dependent DR4 agonism (e.g., Examples 9 and 11). In one aspect, the present invention relates to a multispecific antibody comprising at least (i) a FAPα-binding region capable of binding to FAPα, comprising a first heavy chain variable region and a first light chain variable region, and (ii) a DR4-binding region capable of binding to DR4, comprising a second heavy chain variable region and a second light chain variable region.

[0012] In a further aspect, the present invention relates to a nucleic acid construct, or a combination of nucleic acid constructs, encoding an antibody as defined herein.

[0013] In another aspect, the present invention relates to a composition comprising a nucleic acid construct or a combination of nucleic acid constructs as defined herein.

[0014] In yet another aspect, the present invention relates to a delivery vehicle comprising one or more nucleic acid constructs described herein.

[0015] In yet another aspect, the invention relates to recombinant host cells capable of producing the antibodies described herein, wherein the host cells comprise one or more nucleic acid constructs encoding the antibodies described herein.

[0016] In a still further aspect, the present invention relates to a pharmaceutical composition comprising a multispecific antibody as defined herein and a pharmaceutically acceptable carrier.

[0017] In a still further aspect, the present invention relates to a multispecific antibody as described herein, one or more nucleic acid constructs as described herein, a delivery vehicle as described herein or a pharmaceutical composition as described herein for use in the treatment of cancer.

[0018] In a still further aspect, the present invention relates to a method for producing a multispecific antibody according to the present invention.

[0019] Finally, the present invention also provides monospecific antibodies that target DR4 and FAPα, respectively.

[0020] These and other aspects and embodiments are described in more detail in the following sections. [Brief explanation of the drawings]

[0021] [Figure 1]Binding to human lung fibroblasts and CAFs. (A-B) Binding of BisG1-FAPα-FERL / DR4-FERR, BisG1-FAPα-FERL / b12-FERR, BisG1-b12-FERL / b12-FERR, and IgG1-FAPα-FERL to human lung fibroblasts (A) and CAFs (B) was assessed by flow cytometry. Data shown are geometric mean fluorescence intensity (gMFI) values ​​for one representative experiment out of three. The concentration (μg / mL) at which 50% of the maximal effect (EC50) was observed was derived from the fitted curve. For CAFs (B), the IgG1-FAPα-FERL antibody was included only at the top four concentrations. (C) The binding of two FAPα-targeting antibody clones, FAPα-FEAL and FAP5 (BisG1-FAPα-FEAL / b12-FEAR, BisG1-FAP5-FEAL / b12-FEAR, IgG1-FAPα-FEAL, and IgG1-FAP5-FEAL antibodies), and IgG1-b12-FEAR to human lung fibroblasts was assessed by flow cytometry. Data shown are gMFI values ​​determined by flow cytometry for one representative experiment of two. [Figure 2-1]Binding of DR4-specific antibodies to cell surface-expressed DR4. (A-F) Binding of BisG1-FAPα-FERL / DR4-FERR, BisG1-b12-FERL / DR4-FERR, IgG1-DR4-FERR, and IgG1-b12 was assessed in six different cell lines: colorectal adenocarcinoma cell lines DLD-1 and HCT-15, lung adenocarcinoma A549, colorectal carcinoma HCT-116, breast adenocarcinoma MDA-MB-231, and pancreatic ductal adenocarcinoma PANC-1. Data shown are gMFI values ​​determined by flow cytometry for one representative experiment out of three. (G) DR4 binding of BisG1-b12-FEAL / DR4-T1014A04-FEAR, BisG1-b12-FEAL / DR4-chCTB007-FEAR, IgG1-DR4-chCTB007-FEAR, IgG1-DR4-T1014A04-FEAR, and IgG1-b12-FEAL was assessed in the multiple myeloma cell line OPM-2. Data shown are gMFI values ​​determined by flow cytometry for one experiment. (H) DR4 binding of IgG1-DR4-FERR, BisG1-b12-FERL / DR4-FERR, BisG1-FAPα-FERL / DR4-FERR, IgG1-DR4-chCTB007-FEAR, BisG1-b12-FEAL / DR4-chCTB007-FEAR, IgG1-DR4-T1014A04-FEAR, BisG1-b12-FEAL / DR4-T1014A04-FEAR, and BisG1-b12-FERL / b12-FERR was assessed on the breast adenocarcinoma cell line MDA-MB-231. Data are presented as the mean (±SD) gMFI values ​​determined by flow cytometry of two independent experiments. [Figure 2-2]Binding of DR4-specific antibodies to cell surface-expressed DR4. (A-F) Binding of BisG1-FAPα-FERL / DR4-FERR, BisG1-b12-FERL / DR4-FERR, IgG1-DR4-FERR, and IgG1-b12 was assessed in six different cell lines: colorectal adenocarcinoma cell lines DLD-1 and HCT-15, lung adenocarcinoma A549, colorectal carcinoma HCT-116, breast adenocarcinoma MDA-MB-231, and pancreatic ductal adenocarcinoma PANC-1. Data shown are gMFI values ​​determined by flow cytometry for one representative experiment out of three. (G) DR4 binding of BisG1-b12-FEAL / DR4-T1014A04-FEAR, BisG1-b12-FEAL / DR4-chCTB007-FEAR, IgG1-DR4-chCTB007-FEAR, IgG1-DR4-T1014A04-FEAR, and IgG1-b12-FEAL was assessed in the multiple myeloma cell line OPM-2. Data shown are gMFI values ​​determined by flow cytometry for one experiment. (H) DR4 binding of IgG1-DR4-FERR, BisG1-b12-FERL / DR4-FERR, BisG1-FAPα-FERL / DR4-FERR, IgG1-DR4-chCTB007-FEAR, BisG1-b12-FEAL / DR4-chCTB007-FEAR, IgG1-DR4-T1014A04-FEAR, BisG1-b12-FEAL / DR4-T1014A04-FEAR, and BisG1-b12-FERL / b12-FERR was assessed on the breast adenocarcinoma cell line MDA-MB-231. Data are presented as the mean (±SD) gMFI values ​​determined by flow cytometry of two independent experiments. [Figure 3] Species cross-reactivity to FAPα orthologs. Antibodies BisG1-FAPα-FERL / DR4-FERR, BisG1-FAPα-FEAL / b12-FEAR, IgG1-FAPα-FERL, and IgG1-b12 were tested. (A-F) Binding to Expi293F cells expressing human, cynomolgus monkey, mouse, rat, pig, and dog FAPα, respectively. Data shown are gMFI values ​​determined by flow cytometry for one representative experiment out of three. [Figure 4] Species cross-reactivity to DR4 or mouse / rat DR orthologs. Antibodies BisG1-FAPα-FERL / DR4-FERR, BisG1-DR4-FEAL / b12-FEAR, IgG1-DR4-FERR, and IgG1-b12 were tested. (A) Binding to ExpiCHO-S cells expressing human DR4. (B) Binding to ExpiCHO-S cells expressing cynomolgus monkey DR4. Binding of BisG1-FAPα-FERL / DR4-FERR versus IgG1-DR4-FERR and IgG1-b12 to ExpiCHO-S cells expressing pig DR4 (C), rat DR4 (D), mouse DR4 (E), dog DR4 (F), or rabbit DR4 (G). Data shown are gMFI values ​​determined by flow cytometry for one representative experiment out of three. [Figure 5] Quantification of DR4, DR5, and FAPα surface expression and assessment of fibroblast cell death. (A-B) Surface expression of DR4, DR5, and FAPα in human lung fibroblasts and CAFs. Summary data (mean ± SEM) of two experiments are shown. The horizontal dotted line indicates the lower limit of quantification (LLOQ). (C) Human lung fibroblasts cultured with BisG1-FAPα-FEAL / DR4-FEAR, BisG1-FAPα-FEAL / b12-FEAR, BisG1-b12-FEAL / DR4-FEAR, IgG1-FAPα-FEAL (negative control), or RG7386. The percentage of viable cells normalized to the no-antibody condition is plotted against antibody concentration and shows the mean ± SEM of duplicates. (D) Human lung fibroblasts cultured with 10 μg / mL of BisG1-FAPα-FEAL / DR4-FEAR, BisG1-FAPα-FEAL / b12-FEAR, BisG1-b12-FEAL / DR4-FEAR, or negative control IgG1-b12-FEAR. Staurosporine was used as a positive control. The graph shows the fluorescent signal representing the number of dead cells (duplicates ± SEM) plotted against time. (E) CAFs cultured with BisG1-FAPα-FEAL / DR4-FEAR. The graph shows the % viable tumor cells ± SEM of duplicates, normalized to the no-antibody condition and plotted against antibody concentration. [Figure 6] Transactivation ability of BisG1-FAPα-FEAL / DR4-FEAR. (A) DR4-targeting bispecific antibodies containing FAPα or FAP5 binding arms were used to evaluate DR4 transactivation-mediated targeted cell death in the cancer cell line MDA-MB-231 cocultured with human lung fibroblasts. Negative control antibodies included were BisG1-FAPα-FEAL / b12-FEAR, BisG1-FAP5-FEAL / b12-FEAR, and BisG1-b12-FEAL / DR4-FEAR. 1 μM staurosporine was included as a positive control. Data shown are the mean viable tumor cell surface (μm² / image) ± SEM of duplicates plotted against antibody concentration. (B-C) To evaluate DR4- or DR5-mediated cell death in the absence of fibroblasts, monocultures of MDA-MB-231 (B) or DLD-1 (C) cells were tested using BisG1-FAPα-FEAL / DR4-FEAR, BisG1-β12-FERL / β12-FERR (top four concentrations), or RG7386. (D-E) DR4 transactivation-mediated cell death using BisG1-FAPα-FERL / DR4-FERR, BisG1-β12-FERL / β12-FERR, BisG1-β12-FERL / β12-FERR (top four concentrations), and BisG1-FAPα-FERL / β12-FERR was evaluated in MDA-MB-231 (D) and DLD-1 (E) cancer cell lines cocultured with the FAPα-expressing cell line NIH / 3T3-FAPα. Results in B to D show the mean % viable cells ± SEM of duplicates and are plotted against antibody concentration for one representative experiment out of three. [Figure 7-1]Caspase-8 activation in cocultures of tumor cells with NIH / 3T3-FAPα cells. Caspase-Glo 8 luminescence signals for measurement of caspase-8 activation in cocultures of DLD-1 (A), MDA-MB-231 (C), A549 (E), and SNU-1076 (G) tumor cells with NIH / 3T3-FAPα cells, or in tumor cell monocultures (B, D, F, H), in the presence of BisG1-FAPα-FERL / DR4-FERR, BisG1-b12-FERL / DR4-FERR, or RG7386. Recombinant human TRAIL (the natural ligand for DR4) was included as a positive control. Data presented are the mean ± SEM luminescence (RLU) plotted against antibody concentration from two or three independent experiments. [Figure 7-2] Caspase-8 activation in cocultures of tumor cells with NIH / 3T3-FAPα cells. Caspase-Glo 8 luminescence signals for measurement of caspase-8 activation in cocultures of DLD-1 (A), MDA-MB-231 (C), A549 (E), and SNU-1076 (G) tumor cells with NIH / 3T3-FAPα cells, or in tumor cell monocultures (B, D, F, H), in the presence of BisG1-FAPα-FERL / DR4-FERR, BisG1-b12-FERL / DR4-FERR, or RG7386. Recombinant human TRAIL (the natural ligand for DR4) was included as a positive control. Data presented are the mean ± SEM luminescence (RLU) plotted against antibody concentration from two or three independent experiments. [Figure 8]Targeted cell death of patient-derived organoids (PDOs) via DR4 transactivation in the presence of CAFs. (A-C) The ability of BisG1-FAPα-FEAL / DR4-FEAR and IgG1-FAPα-FEAL to induce DR4 transactivation-mediated cell death of three colorectal cancer (CRC) PDOs was investigated in the presence and absence of CAFs. Data show the survival of PDOs, Hub096 (A), p18T (B), and p19B (C), cultured with (+CAF) or without CAFs in the presence of the indicated antibodies. Cell survival (%) is plotted against antibody concentration (mean ± SEM of duplicates). (D) Apoptosis induction in cocultures of fluorescently labeled Hub096 (CellBrite Blue) and CAFs (CellBrite Orange) in the presence of BisG1-FAPα-FEAL / DR4-FEAR or IgG1-FAPα-FEAL was assessed separately in both cell populations by flow cytometry using Annexin V staining. Data are presented as the mean (±SD) percentage of Annexin V-positive cells from two independent experiments. [Figure 9-1]Evaluation of BisG1-FAPα-FEAL / DR4-FEAR antitumor activity in vivo. (A) FAPα staining in formalin-fixed, paraffin-embedded (FFPE) tissues obtained from PDX models. (A) CTG-1234 (gastric cancer), CTG-1150 (pancreatic cancer), and the positive control (invasive ductal carcinoma) were shown as a percentage of tissue surface area and categorized as 1+ (low) to 3+ (high). (B) Mean (±SEM) tumor volume in the gastric PDX tumor model CTG-1234 after treatment with BisG1-FAPα-FEAL / DR4-FEAR (0.5–8 mg / kg, administered once weekly for 3 weeks) or BisG1-DR4-FEAL / b12-FEAR (8 mg / kg). (C) Kaplan-Meier curves showing progression-free survival (PFS) in CTG-1234 tumor-bearing mice. PFS is defined as the percentage of mice with tumor volumes less than 1000 mm3. (D) Individual tumor volumes and mean ± SEM for each treatment group in the CTG-1234 gastric cancer PDX model on day 42, the final day after all groups were completed. *** = p<0.001 vs. the control BisG1-DR4-FEAL / b12-FEAR treatment group (Mann-Whitney). (E) Mean (± SEM) mouse weights for all treatment groups. (F) Individual tumor volumes and mean ± SEM for each treatment group in the CTG-1150 pancreatic PDX tumor model on day 25, the final day after all groups were completed. * = p<0.05 vs. the control BisG1-DR4-FEAL / b12-FEAR treatment group (Mann-Whitney). (G) Kaplan-Meier curve showing PFS in CTG-1150 tumor-bearing mice, where PFS is defined as the percentage of mice with tumor volumes less than 500 mm3. (H) Mean (±SEM) tumor volume per treatment group over time in the pancreatic PDX tumor model CTG-1150. (I) Mean (±SEM) body weight for all treatment groups. [Figure 9-2]Evaluation of BisG1-FAPα-FEAL / DR4-FEAR antitumor activity in vivo. (A) FAPα staining in formalin-fixed, paraffin-embedded (FFPE) tissues obtained from PDX models. (A) CTG-1234 (gastric cancer), CTG-1150 (pancreatic cancer), and the positive control (invasive ductal carcinoma) were shown as a percentage of tissue surface area and categorized as 1+ (low) to 3+ (high). (B) Mean (±SEM) tumor volume in the gastric PDX tumor model CTG-1234 after treatment with BisG1-FAPα-FEAL / DR4-FEAR (0.5–8 mg / kg, administered once weekly for 3 weeks) or BisG1-DR4-FEAL / b12-FEAR (8 mg / kg). (C) Kaplan-Meier curves showing progression-free survival (PFS) in CTG-1234 tumor-bearing mice. PFS is defined as the percentage of mice with tumor volumes less than 1000 mm3. (D) Individual tumor volumes and mean ± SEM for each treatment group in the CTG-1234 gastric cancer PDX model on day 42, the final day after all groups were completed. *** = p<0.001 vs. the control BisG1-DR4-FEAL / b12-FEAR treatment group (Mann-Whitney). (E) Mean (± SEM) mouse weights for all treatment groups. (F) Individual tumor volumes and mean ± SEM for each treatment group in the CTG-1150 pancreatic PDX tumor model on day 25, the final day after all groups were completed. * = p<0.05 vs. the control BisG1-DR4-FEAL / b12-FEAR treatment group (Mann-Whitney). (G) Kaplan-Meier curve showing PFS in CTG-1150 tumor-bearing mice, where PFS is defined as the percentage of mice with tumor volumes less than 500 mm3. (H) Mean (±SEM) tumor volume per treatment group over time in the pancreatic PDX tumor model CTG-1150. (I) Mean (±SEM) body weight for all treatment groups. [Figure 10]In vivo follow-up evaluation of BisG1-FAPα-FERL / DR4-FERR antitumor activity. Mice bearing established tumors were treated QW × 3 by IV injection with the indicated antibody concentrations (n ​​= 8 mice per group). (A) Mean (±SEM) tumor volume in the gastric PDX tumor model CTG-1234 after treatment with BisG1-b12-FERL / DR4-FERR (2 mg / kg), BisG1-FAPα-FERL / DR4-FERR (0.1, 0.5, or 2 mg / kg), or equimolar doses of RG7386 (0.16, 0.8, or 3.2 mg / kg). (B) Individual tumor volumes and mean ±SEM for each group in the gastric cancer PDX CTG-1234 model on day 12, the final day after all groups were completed. *** = p<0.001 vs. control BisG1-b12-FERL / DR4-FERR treatment group (Mann-Whitney). ** = p<0.01 vs. BisG1-FAPα-FERL / DR4-FERR 0.5 or 2 mg / kg treatment group (Mann-Whitney). (C) Kaplan-Meier curve showing progression-free survival (PFS) of CTG-1234 tumor-bearing mice (PFS is defined as the percentage of mice with tumor volume less than 750 mm3). [Figure 11] Antitumor activity in a multiorgan metastatic mouse model. (A-E) Ex vivo bioluminescence imaging (BioLI) measurements of tumor burden (counts per minute (cpm) / cm², Log10 scale) from each mouse were plotted for each organ and for the indicated treatment group. For statistical analysis, paired t-tests were performed, and p<0.05 was considered statistically significant. Data shown are box plots containing individual data with median values ​​across all animals per treatment group (n=9), including whiskers from minimum to maximum. *p<0.05, **p<0.01. (F) DR4 activation measured by cleaved caspase-3 IHC staining of FFPE sections from resected primary tumors of the cecum and metastatic tissues of the peritoneal wall and liver. The mean percentage (±SEM) of annotated tumor area scored positive for cleaved caspase-3 across all sections analyzed is shown for each treatment group. ns = not significant, ***P ≤ 0.001 ****P ≤ 0.0001 (Mann-Whitney). [Figure 12] Assessment of hepatotoxicity using human liver spheroids. Liver spheroid viability was determined by measuring LDH release (an indicator of plasma membrane damage) after 4 days of culture with BisG1-FAPα-FEAL / DR4-FEAR, ABBV-621-Fc fusion, RG7386, IgG1-b12-FEAR, or IgG1-b12 antibody (A and C), and intracellular ATP levels (an indicator of metabolically active cells) after 6 days (B) or 7 days (D) of culture. The dashed line represents the lower limit of detection (LLOD) for LDH. Data shown are the mean ± SEM of four technical replicates per condition of bioluminescence signal. [Figure 13] Cytotoxicity in cocultures of tumor cells with NIH / 3T3-FAPα cells at different ratios. CellTiter-Glo viability data for (A) DLD-1 or (B) MDA-MB-231 tumor cells cocultured with NIH / 3T3-FAPα cells at different ratios of tumor cells to NIH / 3T3-FAPα cells in the presence of BisG1-FAPα-FERL / DR4-FERR. Data are shown as the mean (±SD) % viable cells plotted against antibody concentration for duplicates from one representative of three independent experiments. [Figure 14] Cytotoxicity in PDO cocultures with FAPα knockdown CAFs. Cocultures of CRC-derived PDO (A) Tor9, (B) p19B, (C) Hub096, and (D) Hub098 CAFs transduced with FAPα shRNA (CAF#34) or non-targeting shRNA (CAF#Scr) were treated with BisG1-FAPα-FERL / DR4-FERR or negative control antibody BisG1-b12-FERL / b12-FERR. CellTiter-Glo survival data are shown as mean ± SD % cell survival plotted against antibody concentration for one experiment. [Figure 15]Cytotoxicity and DR4 transactivation in the presence of soluble FAPα. CellTiter-Glo cytotoxicity data for monocultures of (A) DLD-1 and (B) MDA-MB-231 tumor cell lines in the presence of soluble FAPα (29.4 nM) and BisG1-FAPα-FERL / DR4-FERR. Data are shown as mean ± SEM % viable cells plotted against antibody concentration for two to three independent experiments. [Figure 16] Effect on FAPα enzymatic activity. Fluorogenic dipeptidyl peptidase substrate was incubated with BisG1-FAPα-FERL / DR4-FERR, BisG1-b12-FERL / b12-FERR, or the positive control chemical dipeptidyl peptidase inhibitor Talabostat in the presence of recombinant human FAPα. Fluorescent 7-amino-4-methylcoumarin (AMC) was measured as a readout of FAPα dipeptidyl peptidase activity. Data shown are AMC concentrations plotted against antibody concentrations for one representative of two experiments. [Figure 17] C1q binding to cell membrane-bound BisG1-FAPα-FERL / DR4-FERR. (A) Binding of BisG1-FAPα-FERL / DR4-FERR and the IgG1-b12-FER nonbinding control antibody to MDA-MB-231 cells, and (B) C1q binding to cell surface-bound BisG1-FAPα-FERL / DR4-FERR, were determined by flow cytometry using MDA-MB-231 cells in the presence of 20% NHS. Data shown are gMFI values ​​determined by flow cytometry for one representative experiment out of three independent experiments. [Figure 18] Binding of BisG1-FAPα-FERL / DR4-FERR to immobilized FcγRs. Binding of BisG1-FAPα-FERL / DR4-FERR to immobilized recombinant human FcγRs (FcγRIa, FcγRIIa-H131, FcγRIIa-R131, FcγRIIb, FcγRIIIa-F158, FcγRIIIa-V158) was analyzed by SPR. For one experiment, the relative binding response is plotted against the antibody concentration. [Figure 19] Binding of BisG1-FAPα-FERL / DR4-FERR to immobilized FcRn. Binding of BisG1-FAPα-FERL / DR4-FERR to immobilized recombinant human FcRn at pH 6.0 (A) and pH 7.4 (B) was analyzed by SPR. Sensorgrams show raw data from one representative run of two (pH 6.0) or four (pH 7.4) runs as dashed lines, and curve fitting as solid black lines. [Figure 20] Pharmacokinetic profile of BisG1-FAPα-FERL / DR4-FERR in non-tumor-bearing mice. Mice were injected IV with a single dose of 2 mg / kg BisG1-FAPα-FERL / DR4-FERR, and total human IgG concentrations in plasma samples were determined by ECLIA. Data shown are the mean plasma IgG concentrations over time after treatment in C57BL / 6 SCID mice (n=1) and hFcRn SCID mice (n=3). The 95% confidence interval for the pharmacokinetic profile of wild-type IgG1 in hFcRn SCID mice is indicated by the gray shaded area. DETAILED DESCRIPTION OF THE INVENTION

[0022] definition The term "antibody," as used herein, is intended to refer to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen under typical physiological and / or tumor-specific conditions for a significant period of time, e.g., a half-life of at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours or more, at least about 48 hours or more, at least about 3, 4, 5, 6, 7 days or more, or any other relevant, functionally defined period of time, e.g., a time sufficient to induce, promote, enhance, and / or modulate a physiological response associated with antibody binding to the antigen. The binding region (or binding domain, as may be used herein, both have the same meaning) that interacts with the antigen includes the variable regions of both the heavy and light chains of the immunoglobulin molecule. The antibody constant region (Ab) can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system, such as C1q, the first component in the classical pathway of complement activation. Alternatively, antibody constant regions may be silenced, for example, by mutation, such that they are unable to activate the complement system, or at least less efficiently. The term "antibody" includes antibody-like polypeptides, such as monoclonal antibodies (mAbs), chimeric antibodies, and humanized antibodies, that retain the ability to specifically bind to an antigen (antigen-binding fragment) and be conjugated to a toxin, provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant technology, as well as "antibody fragments" or "fragments thereof." Antibodies defined in accordance with the present invention can have any isotype, unless otherwise limited by the disclosure herein. As noted above, the term antibody, as used herein, includes fragments of antibodies that retain the ability to specifically interact with, e.g., bind to, an antigen, unless otherwise specified or clearly contradicted by the context. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed by the term "antibody" include (i) Fab' or Fab fragments, monovalent fragments consisting of a light chain variable domain (VL), a heavy chain variable domain (VH), a light chain constant region (CL) and heavy chain constant region domain 1 (CH1) domains, or the monovalent antibodies described in WO 2007 / 059782; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting essentially of a VH domain and a CH1 domain; (iv) Fv fragments consisting essentially of the VL and VH domains of a single arm of an antibody; (v) dAb fragments consisting essentially of a VH domain, also called domain antibodies (Holt et al; Trends Biotechnol. 2003 Nov;21(11):484-90), dAb fragments (Ward et al., Nature 341, 544-546 (1989)), (vi) camelids or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5(1): 111-24), and (vii) isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they may be linked by a synthetic linker using recombinant techniques, allowing the VL and VH domains to be paired together to form a single protein chain that forms a monovalent molecule (known as a single-chain antibody or single-chain Fv (scFv)). See, for example, Revets et al; Expert Opin Biol Ther. 2005 Jan; 5(1): 111-24, and Bird et al., Science 242, 423-426 (1988). Such single chain antibodies are encompassed within the term antibody unless otherwise specified or clearly indicated by the context. Although such fragments are generally included within the meaning of antibody, they collectively and each independently represent a unique feature of the present invention, exhibiting different biological properties and utilities. These and other useful antibody fragments in the context of the present invention are discussed further herein.Antibodies can be produced and recovered from different in vitro or ex vivo expression or production systems, such as recombinant modified host cells, from hybridomas or systems using cell extracts that support the in vitro transcription and / or translation of nucleic acid sequences encoding the antibodies. It should be understood that a multitude of different antibodies, as defined in the context of the present invention, can be provided by producing each antibody separately in a production system as described above and then mixing the antibodies, or by producing several antibodies in the same production system.

[0023] As used herein, the term "immunoglobulin heavy chain" or "heavy chain of an immunoglobulin" is intended to refer to one of the heavy chains of an immunoglobulin. A heavy chain is typically composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH), which define the immunoglobulin isotype. The heavy chain constant region is typically composed of three domains, CH1, CH2, and CH3. As used herein, the term "immunoglobulin" is intended to refer to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains: one pair of light (L) low-molecular-weight chains and one pair of heavy (H) chains, all four potentially interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized (see, e.g., Fundamental Immunology Ch. 7 (Paul, W., 2nd ed. Raven Press, NY (1989). Within the immunoglobulin structure, two heavy chains are interconnected via disulfide bonds at the so-called "hinge region." Like heavy chains, each light chain typically consists of several regions: a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically consists of one domain, CL. Furthermore, the VH and VL regions can be further subdivided into regions of hypervariability (or hypervariable regions that may be hypervariable in sequence and / or in the form of structurally defined loops), also called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDR sequences are determined using the IMGT (Lefranc MP. et al., Nucleic Acids Research, 27, 209-212, 1999] and Brochet X. Nucl. Acids Res. 36, W503-508 (2008)).

[0024] As used herein, the terms "half molecule," "Fab arm," and "arm" refer to one heavy-light chain pair. When a bispecific antibody is described as comprising a half molecule antibody "derived from" a first antibody and a half molecule antibody "derived from" a second antibody, the term "derived from" indicates that the bispecific antibody was produced by recombining, by any known method, the half molecules from each of the first and second antibodies into the resulting bispecific antibody. In this context, "recombinant" is not intended to be limited by any particular recombination method and thus includes all of the methods for producing bispecific antibodies described herein below, including, for example, recombination by half molecule exchange, as well as recombination at the nucleic acid level and / or recombination by co-expression of two half molecules in the same cell.

[0025] As used herein, the term "antigen-binding region" or "binding region" refers to the region of an antibody that can bind to an antigen and contains an epitope. An antigen can be any molecule, such as a polypeptide present on a cell, bacterium, or virion. The terms "antigen" and "target" can be used interchangeably in the context of the present invention, unless the context indicates otherwise. The terms "antigen-binding region" and "antigen-binding site" can be used interchangeably in the context of the present invention, unless the context indicates otherwise.

[0026] The term "epitope" refers to an antigenic determinant that is specifically bound by an antibody. Epitopes usually consist of surface groupings of molecules, such as amino acids, sugar side chains, or combinations thereof, and usually have specific three-dimensional structural and charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former, but not the latter, is lost in the presence of denaturing solvents. Epitopes can include amino acid residues that are directly involved in binding as well as other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked or covered by an antibody when bound to an antigen (in other words, amino acid residues within or close to the footprint of a specific antibody).

[0027] The antibody binding region can be determined by epitope binning using biolayer interferometry, by alanine scanning, or by shuffle assays (using antigen constructs in which regions of the antigen are exchanged with regions of another species and determining whether the antibody still binds to the antigen). Amino acids within the antibody binding region involved in interactions with the antibody can be determined by hydrogen / deuterium exchange mass spectrometry and crystallography of the antibody bound to the antigen.

[0028] The terms "blocking binding" or "blocking antibody binding" or "cross-blocking binding" or "cross-blocking binding" refer to a situation in which one antibody bound to a specific antigen prevents another antibody from binding to the same antigen. In the absence of the other antibody, each antibody has the ability to bind to the antigen as determined by a significant binding response, but in the presence of the other antibody, one of the antibodies lacks a binding response. This type of behavior indicates that both antibodies bind to substantially overlapping epitopes on the antigen. The ability of one antibody to block the binding of another antibody can be determined by biolayer interferometry in a classic sandwich epitope binning assay format, for example, as described in Abdiche et al. (Abdiche YN, Malashock DS, Pinkerton A, Pons J. Exploring blocking assays using Octet, ProteOn, and Biacore biosensors. Anal Biochem. 2009;386(2):172-180). Briefly, in sandwich epitope binning assay, antibody in solution is tested for binding to its specific antigen, which is first captured by immobilized antibody.In the context of the present invention, one antibody does not block the binding of other antibodies if it can "displace" other antibodies, that is, one antibody dissociates from the antigen when other antibodies bind to the antigen.The terms "blocking binding" and "blocking antibody binding" and "cross-blocking binding" and "cross-blocking binding" can be used interchangeably in the context of the present invention, unless the context is inconsistent.Preferably, the ability of one antibody to block the binding of another antibody is determined using full-length antibodies.

[0029] As used herein, the term "binding" refers to binding that is typically 1E, as determined by biolayer interferometry. -6 M or less, for example, 5E -7 M or less, 1E -7 M or less, for example, 5E -8 M or less, for example, 1E -8M or less, for example, 5E -9 M or less, for example, 1E -9 M or less, for example, 5E -10 M or less, for example, 1E -10 M or less, for example, 5E -11 M or less, for example, 1E -11 M or less, for example, 5E -12 M or less, or for example 1E -12 K below M D It refers to the binding of an antibody to a predetermined antigen or target with a binding affinity equivalent to

[0030] As used herein, "K" D The term "(M)" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, k d k a It is obtained by dividing by

[0031] As used herein, "k" d " (sec -1 The term k ) refers to the dissociation rate constant of a particular antibody-antigen interaction. off Also called value or off-rate.

[0032] As used herein, "k" a " (M -1 x sec -1 The term k ) refers to the association rate constant of a particular antibody-antigen interaction. on Also called value or on-speed.

[0033] As used herein, the term "cis binding" refers to the simultaneous binding of a multispecific antibody to different targets on the same cell. For example, a bispecific antibody can bind to its two targets on the same cell.

[0034] As used herein, the term "trans-binding" refers to the simultaneous binding of a multispecific antibody to different targets on different cells. For example, a bispecific antibody can bind to two cells by binding to one target on a first cell and another target on a second cell.

[0035] As used herein, the term "FAPα" refers to a protein titled fibroblast activation protein α, which is an enzyme encoded by the FAP gene and is also known as surface-expressed protease (seprase), serine integral membrane protease (SIMP), dipeptidyl peptidase FAP, prolyl endopeptidase FAP, and integral membrane serine protease. It is a 170 kDa transmembrane protein. In humans (Homo sapiens), the FAPα protein has the amino acid sequence set forth in SEQ ID NO: 33 ([prolyl endopeptidase FAP]: Uniprot accession number [Q12884]). In the amino acid sequence set forth in SEQ ID NO: 33, amino acid residues [1-4] are a cytoplasmic peptide, amino acid residues [5-25] are a transmembrane peptide, and amino acid residues [26-760] are an extracellular polypeptide. In cynomolgus monkeys (Macaca fascicularis), the FAPα protein has the amino acid sequence set forth in SEQ ID NO: 39 (Uniprot Accession No. A0A2K5VGF4), where amino acid residues [1-760] are the mature polypeptide. In mice (Mus musculus), the FAPα protein has the amino acid sequence set forth in SEQ ID NO: 35 (Uniprot Accession No. P97321), where amino acid residues [1-761] are the mature polypeptide. In rats (Rattus norvegicus), the FAPα protein has the amino acid sequence set forth in SEQ ID NO: 36 (Uniprot Accession No. Q8R492), where amino acid residues [1-761] are the mature polypeptide. In dogs (Canis lupus familiaris), the FAPα protein has the amino acid sequence set forth in SEQ ID NO: 37 (Uniprot Accession No. A0A8C0NKP1), where amino acid residues [1-760] are the mature polypeptide. In pigs (Sus scrofa), the FAPα protein has the amino acid sequence shown in SEQ ID NO: 38 (Uniprot accession number K7GQN2), with amino acid residues [1-760] being the mature polypeptide.

[0036] The term "DR4" as used herein refers to a protein entitled death receptor 4, which is a receptor for the cytotoxic ligand TNFSF10 / TRAIL and is also known as tumor necrosis factor receptor superfamily member 10A, APO2, CD261, and TNF-related apoptosis-inducing ligand receptor 1 (TRAIL receptor 1; TRAIL-R1). It is a 56-kDa transmembrane protein. In humans (Homo sapiens), the DR4 protein has the amino acid sequence set forth in SEQ ID NO: 68 (tumor necrosis factor receptor superfamily member 10A). In the amino acid sequence set forth in SEQ ID NO: 68, amino acid residues [1-23] are the signal peptide, and amino acid residues [24-468] are the mature polypeptide. In cynomolgus monkeys (Macaca fascicularis), the DR4 protein has the amino acid sequence set forth in SEQ ID NO: 69. In the amino acid sequence shown in SEQ ID NO: 69, amino acid residues [1-23] are the signal peptide, and amino acid residues [24-471] are the mature polypeptide.

[0037] The term "CAF" as used herein refers to cancer-associated fibroblasts, a heterogeneous population of stromal cells with mesenchymal lineage that reside in the tumor microenvironment and coexist with the growing tumor mass. CAFs are spindle-shaped cells that build and remodel extracellular matrix structures. The definition of CAFs is based on a combination of morphological characteristics, biomarkers, and genetic mutations.

[0038] As used herein, the term "tumor microenvironment" or "TME" refers to the ecosystem surrounding a tumor in the body. The TME is a complex and dynamic environment that influences tumor growth, invasion, and metastasis. The tumor and TME constantly interact and influence each other both positively and negatively. The TME includes immune cells, extracellular matrix, blood vessels, and stromal cells.

[0039] As used herein, the terms "monoclonal antibody," "monoclonal Ab," "monoclonal antibody composition," "mAb," and the like refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity having variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced by hybridomas comprising B cells obtained from a transgenic or transchromosomal non-human animal, e.g., a transgenic mouse, whose genome contains human heavy and light chain transgenes, fused to an immortalized cell. Monoclonal antibodies can also be produced from systems using recombinantly modified host cells or cell extracts that support in vitro transcription and / or translation of nucleic acid sequences encoding the antibody.

[0040] As used herein, the term "isotype" refers to the immunoglobulin class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or any allotype thereof, e.g., IgG1m(za) and IgG1m(f)), encoded by heavy chain constant region genes. Furthermore, each heavy chain isotype can be combined with either a kappa (κ) or lambda (λ) light chain.

[0041] The term "allotype" as used herein refers to amino acid variation within an isotype class of the same species. The major allotypes of antibody isotypes vary between ethnic individuals. Known allotypic variation within the IgG1 isotype of the heavy chain results from four amino acid substitutions within the antibody framework. In one embodiment, the antibody of the present invention is of the IgG1m(f) allotype as defined by SEQ ID NO: 21. In one embodiment of the present invention, the first and second antibodies of the present invention are of the IgG1m(f) allotype as defined by SEQ ID NO: 21, with at least one amino acid substitution introduced. In one embodiment of the present invention, the first and second antibodies of the present invention are of the IgG1m(f) allotype as defined by SEQ ID NO: 21, with up to five amino acid substitutions introduced, for example, four amino acid substitutions, for example, three amino acid substitutions, for example, two amino acid substitutions.

[0042] The term "full-length," when used in the context of an antibody, indicates that the antibody is not a fragment and contains all of the domains of a particular isotype normally found in that isotype in nature, e.g., the VH, CH1, CH2, CH3, hinge, VL, and CL domains of an IgG1 antibody. In some embodiments, the term "full-length," when used in the context of an antibody, refers to an antibody (e.g., a parent antibody or a variant antibody) that contains one or two pairs of heavy and light chains, each containing all the heavy and light chain constant and variable domains normally found in a heavy-light chain pair in a wild-type antibody of that isotype. In a full-length variant antibody, the heavy and light chain constant and variable domains may contain amino acid substitutions that improve the functional properties of the antibody when compared to the full-length parent or wild-type antibody. These include substitutions to reduce antibody effector function and to facilitate the construction of multispecific antibodies, such as bispecific antibodies. Full-length antibodies according to the present invention can be produced by a method comprising (i) cloning the CDR sequences into a suitable vector containing the complete heavy and light chain sequences, and (ii) expressing the complete heavy and light chain sequences in a suitable expression system. It is within the knowledge of one skilled in the art to produce full-length antibodies starting from either the CDR sequences or the complete variable region sequences.

[0043] As used herein, the term "human antibody" is intended to include antibodies having variable and framework regions derived from human germline immunoglobulin sequences and human immunoglobulin constant domains. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions, or deletions introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another non-human species, such as a mouse, have been grafted onto human framework sequences.

[0044] As used herein, the term "Fc-mediated effector function" is intended to refer to a function that results in the binding of a polypeptide or antibody to its target or antigen on a cell membrane, where an Fc effector function is attributable to the Fc region of a polypeptide or antibody. Examples of Fc effector functions include (i) C1q binding, (ii) complement activation, (iii) complement-dependent cytotoxicity (CDC), (iv) antibody-dependent cell-mediated cytotoxicity (ADCC), (v) Fc-gamma receptor binding, (vi) antibody-dependent cellular phagocytosis (ADCP), (vii) complement-dependent cytotoxicity (CDCC), (viii) complement-enhanced cytotoxicity, (ix) antibody-mediated opsonization, binding of an antibody to a complement receptor, (x) opsonization, and (xi) any combination of (i)-(x).

[0045] The term "hinge region" as used herein refers to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 according to the EU numbering system set forth in Kabat (Kabat, E.A. et al., Sequences of proteins of immunological interest. 5th Edition - U.S. Department of Health and Human Services, NIH Publication No. 91-3242, pp. 662, 680, 689 (1991)). However, the hinge region may be of any of the other subtypes described herein.

[0046] As used herein, the term "CH1 region" or "CH1 domain" refers to the CH1 region of an immunoglobulin heavy chain. Thus, for example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118-215 according to the Eu numbering system as set forth in Kabat (ibid.). However, the CH1 region may be of any of the other subtypes described herein.

[0047] As used herein, the term "CH2 region" or "CH2 domain" refers to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the Eu numbering system as set forth in Kabat (ibid.). However, the CH2 region may be of any of the other subtypes described herein.

[0048] As used herein, the term "CH3 region" or "CH3 domain" refers to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the Eu numbering system as set forth in Kabat (ibid.). However, the CH3 region may be of any of the other subtypes described herein.

[0049] As used herein, the terms "inactive," "inactive," or "non-activating" refer to an Fc region that is, at a minimum, unable to bind to any Fc gamma receptor (FcgR), unable to induce Fc-mediated cross-linking of FcgR, or unable to induce FcgR-mediated cross-linking of target antigens via the two Fc regions of an individual antibody, or unable to bind C1q. The inactivity of an antibody Fc region can be tested using antibodies in monospecific or bispecific formats.

[0050] The term "monovalent antibody," in the context of the present invention, refers to an antibody molecule that can interact with a specific epitope on an antigen via only one antigen-binding domain (e.g., one Fab arm). In the context of a bispecific antibody, "monovalent antibody binding" refers to the binding of the bispecific antibody to one specific epitope on an antigen via only one antigen-binding domain (e.g., one Fab arm).

[0051] The term "monospecific antibody" in the context of the present invention refers to an antibody that has binding specificity for only one epitope. An antibody can be a monospecific monovalent antibody (i.e., having only one antigen-binding region) or a monospecific bivalent antibody (i.e., having two identical antigen-binding regions). Thus, an antibody can be a monospecific antibody with monovalent binding (i.e., having only one antigen-binding region) or a monospecific antibody with bivalent binding (i.e., having two identical antigen-binding regions).

[0052] The term "bispecific antibody" refers to an antibody having two non-identical antigen-binding domains, such as two non-identical Fab arms or two Fab arms with non-identical CDR regions. In the context of the present invention, a bispecific antibody has specificity for at least two different epitopes. Such epitopes can be on the same or different antigens or targets. When the epitopes are on different antigens, such antigens can be on the same cell or different cells, cell types, or structures, such as extracellular matrix or vesicles and soluble proteins. Thus, a bispecific antibody can cross-link multiple antigens, such as two different cells.

[0053] The term "bivalent antibody" refers to an antibody having two antigen-binding regions that bind to epitopes on one or two targets or antigens, or to one or two epitopes on the same antigen. Thus, a bivalent antibody can be a monospecific bivalent antibody or a bispecific bivalent antibody; i.e., a bivalent antibody can be a monospecific antibody with bivalent binding or a bispecific antibody with bivalent binding. In one embodiment, a bispecific antibody with bivalent binding is a bispecific antibody with monovalent binding to a first target and monovalent binding to a second target.

[0054] The term "multispecific antibody" refers to an antibody having two or more non-identical antigen-binding domains, such as two or more non-identical Fab arms or two or more Fab arms with non-identical CDR regions. In the context of the present invention, a multispecific antibody has specificity for at least two different epitopes. Such epitopes can be on the same or different antigens or targets. When the epitopes are on different antigens, such antigens can be on the same cell or different cells, cell types, or structures, such as extracellular matrix or vesicles and soluble proteins. Thus, a multispecific antibody can crosslink multiple antigens, such as two different cells.

[0055] The terms "amino acid" and "amino acid residue" may be used interchangeably herein and should not be understood as limiting. Amino acids are organic compounds containing an amine (-NH2) and a carboxyl (-COOH) functional group, along with a side chain (R group) specific to each amino acid. In the context of the present invention, amino acids may be classified based on their structure and chemical properties. Thus, the classes of amino acids may be reflected in one or both of the following tables:

[0056] [Table 1]

[0057] [Table 2]

[0058] The substitution of one amino acid for another amino acid can be classified as conservative substitution or non-conservative substitution.In the context of the present invention, "conservative substitution" refers to the substitution of one amino acid with another amino acid that has similar structural and / or chemical characteristics, and such substitution of one amino acid residue is the substitution of another amino acid residue of the same class as defined in either of the two tables above.For example, leucine can be substituted with isoleucine, because both are aliphatic branched hydrophobic residues.Similarly, aspartic acid can be substituted with glutamic acid, because both are small and negatively charged residues.

[0059] In the context of the present invention, substitutions in antibodies are indicated as follows:

[0060] Original amino acid - position - replacement amino acid Referring to the well-recognized nomenclature for amino acids, three-letter or one-letter codes are used, including the code "Xaa" or "X" to indicate any amino acid residue. Thus, Xaa or X can typically represent any of the 20 naturally occurring amino acids. As used herein, the term "naturally occurring" refers to any one of the following amino acid residues: glycine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, asparagine, glutamic acid, glutamine, proline, tryptophan, phenylalanine, tyrosine, methionine, and cysteine. Thus, the designation "K409R" or "Lys409Arg" means that the antibody contains a substitution of lysine with arginine at amino acid position 409.

[0061] Substitution of an amino acid at a given position with any other amino acid is The original amino acid position is referred to as "K409", for example.

[0062] In the case of modifications in which the original and / or replacement amino acids may include multiple, but not all, amino acids, the multiple amino acids are separated by "," or " / ." For example, substitution of lysine with arginine, alanine, or phenylalanine at position 409 is "Lys409Arg, Ala, Phe" or "Lys409Arg / Ala / Phe" or "K409R, A, F" or "K409R / A / F" or "K409 to R, A or F".

[0063] Such designations may be used interchangeably in the context of this invention and have the same meaning and purpose.

[0064] Furthermore, the term "substitution" encompasses substitution with any one or other of the 19 naturally occurring amino acids, or with other amino acids, such as unnatural amino acids. For example, substitution of amino acid K at position 409 includes each of the substitutions 409A, 409C, 409D, 409E, 409F, 409G, 409H, 409I, 409L, 409M, 409N, 409Q, 409R, 409S, 409T, 409V, 409W, 409P, and 409Y. This is equivalent to the symbol 409X, where X represents any amino acid other than the original amino acid. These substitutions may also be referred to as K409A, K409C, etc., or K409A, C, etc., or K409A / C / etc. The same applies equally to each and every position referred to herein, and any one of such substitutions is specifically included herein.

[0065] As used herein, the term "amino acid corresponding to position ..." refers to the amino acid position number in the human IgG1 heavy chain. Corresponding amino acid positions in other immunoglobulins can be found by alignment with human IgG1. Thus, an amino acid or segment in one sequence that "corresponds" to an amino acid or segment in another sequence is one that aligns with the other amino acid or segment using standard sequence alignment programs. Methods for aligning sequences or segments in sequences, thereby determining positions in the sequences that correspond to amino acid positions according to the present invention, are believed to be well known in the art.

[0066] As used herein, the term "host cell" is intended to refer to a cell into which an expression vector has been introduced. It should be understood that such a term is intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, transfectomas such as CHO cells, HEK-293 cells, Expi293F cells, PER.C6 cells, NS0 cells, and lymphocytic cells, as well as prokaryotic cells such as E. coli and other eukaryotic hosts such as plant cells and fungi.

[0067] As used herein, the term "transfectoma" includes recombinant eukaryotic host cells, such as CHO cells, PER.C6 cells, NS0 cells, HEK-293 cells, Expi293F cells, plant cells, or fungi, including yeast cells, that express an antibody or target antigen.

[0068] For purposes of the present invention, "sequence identity" between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) as implemented in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably the Needle program version 5.0.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

[0069] (identical residues × 100) / (length of alignment − total number of gaps in the alignment) Retention of similar residues may also, or alternatively, be measured by a similarity score determined by use of a BLAST program (e.g., BLAST 2.2.8 available from NCBI using standard settings: BLOSUM62, Open Gap=11, Extended Gap=1). Suitable variants typically exhibit at least about 45%, e.g., at least about 55%, at least about 65%, at least about 75%, at least about 85%, at least about 90%, at least about 95%, or more (e.g., about 99%) similarity to the parent sequence.

[0070] As used herein, the term "internalized" or "internalization" refers to the biological process by which a molecule, such as an antibody according to the present invention, is engulfed by the cell membrane and drawn into the interior of the cell. Internalization is sometimes also called "endocytosis."

[0071] multispecific antibodies In a first aspect, the present invention relates to a multispecific antibody comprising at least (i) a FAPα-binding region comprising a first heavy chain variable region and a first light chain variable region, and (ii) a DR4-binding region comprising a second heavy chain variable region and a second light chain variable region. In one embodiment, the FAPα-binding region is capable of binding to FAPα. In a further embodiment, the DR4-binding region is capable of binding to DR4.

[0072] In a further aspect, the present invention relates to a multispecific antibody comprising at least (i) a FAPα-binding region capable of binding to FAPα comprising a first heavy chain variable region and a first light chain variable region, and (ii) a DR4-binding region capable of binding to DR4 comprising a second heavy chain variable region and a second light chain variable region.

[0073] The present invention further provides a multispecific antibody as described herein, wherein the multispecific antibody is a bispecific antibody with monovalent binding to FAPα and monovalent binding to DR4. In a further aspect, the present invention relates to a bispecific antibody with monovalent binding to FAPα (e.g., one Fab arm that binds to FAPα) and monovalent binding to DR4 (e.g., one Fab arm that binds to DR4).

[0074] As known to those skilled in the art, each antigen-binding region of an antibody generally comprises a heavy chain variable region (VH) and a light chain variable region (VL), each of which comprises three CDR sequences, CDR1, CDR2, and CDR3, respectively, and may comprise four framework sequences, FR1, FR2, FR3, and FR4, respectively. This structure is also preferably found in antibodies according to the present invention. In one embodiment, one, two, three, or all of the four framework sequences are human framework sequences. The CDR1, CDR2, and CDR3 regions can be identified from the variable heavy chain region and the variable light chain region using methods known in the art.

[0075] The FAPα-binding region of a multispecific antibody may comprise a heavy chain variable region (VH) comprising three complementarity determining regions, CDR1, CDR2 and CDR3, present within the amino acid sequence set forth in SEQ ID NO: 13. The FAPα-binding region of a multispecific antibody may also comprise a light chain variable region (VL) comprising three complementarity determining regions, CDR1, CDR2 and CDR3, present within the amino acid sequence set forth in SEQ ID NO: 14. In one embodiment, the FAPα-binding region of a multispecific antibody described herein comprises a heavy chain variable region (VH) comprising three complementarity determining regions, CDR1, CDR2 and CDR3, present within the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable region (VL) comprising three complementarity determining regions, CDR1, CDR2 and CDR3, present within the amino acid sequence set forth in SEQ ID NO: 14.

[0076] Further disclosed herein is a multispecific antibody, wherein the FAPα-binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 1, 2, and 3, respectively. Also disclosed herein is a multispecific antibody, wherein the FAPα-binding region comprises a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 4, 5, and 6, respectively. In one embodiment, the FAPα-binding region of the multispecific antibody comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 1, 2, and 3, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 4, 5, and 6, respectively. The CDR regions from the variable heavy and light chain regions are annotated according to IMGT (see Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, Developmental and Comparative Immunology, 27(1), 55-77 (2003)).

[0077] The present disclosure further provides multispecific antibodies, wherein the VH sequence of the FAPα-binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. The present disclosure also provides multispecific antibodies, wherein the VL sequence of the FAPα-binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In further embodiments, the VH sequence of the FAPα-binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 13, and said VL sequence of the FAPα-binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 14.

[0078] Furthermore, the multispecific antibody may further comprise a framework region of the VH sequence of the FAPα-binding region that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. Furthermore, the multispecific antibody may further comprise a framework region of the VL sequence of the FAPα-binding region that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In a further embodiment, the framework regions of the VH sequence of the FAPα-binding region have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 13, and the framework regions of the VL sequence of the FAPα-binding region have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 14.

[0079] In further embodiments, the framework regions of the VH sequence of the FAPα-binding region of a multispecific antibody described herein have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the framework regions of the amino acid sequence set forth in SEQ ID NO: 13. In still further embodiments, the framework regions of the VL sequence of the FAPα-binding region of a multispecific antibody described herein have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the framework regions of the amino acid sequence set forth in SEQ ID NO: 14. In yet a further embodiment, the framework regions of the VH sequence of the FAPα-binding region have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the framework regions of the amino acid sequence set forth in SEQ ID NO: 13, and the framework regions of the VL sequence of the FAPα-binding region have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the framework regions of the amino acid sequence set forth in SEQ ID NO: 14.

[0080] In yet one embodiment, the multispecific antibody comprises a VH sequence of the FAPα-binding region that has, outside of the CDR regions, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In a further embodiment, the multispecific antibody comprises a VL sequence of the FAPα-binding region that has, outside of the CDR regions, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In a further embodiment, the multispecific antibody comprises a VH sequence of the FAPα-binding region that has, outside the CDR regions, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 13, and a VL sequence of the FAPα-binding region that has, outside the CFR regions, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 14.

[0081] In a still further embodiment, said VH sequence of the FAPα-binding region as set forth in SEQ ID NO: 13 comprises up to 10 substitutions, such as up to 9 substitutions, for example up to 8 substitutions, such as up to 7 substitutions, for example up to 6 substitutions, such as up to 5 substitutions, for example up to 4 substitutions, such as up to 3 substitutions, for example up to 2 substitutions, such as up to 1 substitution. In a still further embodiment, said VL sequence of the FAPα-binding region as set forth in SEQ ID NO: 14 comprises up to 10 substitutions, such as up to 9 substitutions, for example up to 8 substitutions, such as up to 7 substitutions, for example up to 6 substitutions, such as up to 5 substitutions, for example up to 4 substitutions, for example up to 3 substitutions, such as up to 2 substitutions, for example up to 1 substitution.

[0082] Alternatively, said VH sequence of the FAPα binding region differs from SEQ ID NO: 13 by up to 10 substitutions, such as up to 9 substitutions, for example up to 8 substitutions, such as up to 7 substitutions, for example up to 6 substitutions, such as up to 5 substitutions, for example up to 4 substitutions, such as up to 3 substitutions, for example up to 2 substitutions, such as up to 1 substitution. In a further embodiment, said VL sequence of the FAPα binding region differs from SEQ ID NO: 14 by up to 10 substitutions, such as up to 9 substitutions, for example up to 8 substitutions, such as up to 7 substitutions, for example up to 6 substitutions, such as up to 5 substitutions, for example up to 4 substitutions, such as up to 3 substitutions, for example up to 2 substitutions, such as up to 1 substitution.

[0083] In a still further embodiment, said VH sequence of the FAPα binding region set forth in SEQ ID NO: 13 comprises 1 to 10 substitutions, such as 1 to 9 substitutions, for example 1 to 8 substitutions, such as 1 to 7 substitutions, for example 1 to 6 substitutions, such as 1 to 5 substitutions, for example 1 to 4 substitutions, such as 1 to 3 substitutions, for example 1 to 2 substitutions. In a still further embodiment, said VL sequence of the FAPα binding region set forth in SEQ ID NO: 14 comprises 1 to 10 substitutions, such as 1 to 9 substitutions, for example 1 to 8 substitutions, such as 1 to 7 substitutions, for example 1 to 6 substitutions, for example 1 to 5 substitutions, for example 1 to 4 substitutions, such as 1 to 3 substitutions, for example 1 to 2 substitutions.

[0084] In still further embodiments, the VH and VL sequences of the FAPα-binding domains differ only in the framework regions. In one embodiment, in the VH sequence according to SEQ ID NO: 13, FR1 is defined by amino acid residues 1-25, FR2 is defined by amino acid residues 34-50, FR3 is defined by amino acid residues 58-96, and FR4 is defined by amino acid residues 111-121. In another embodiment, in the VL sequence according to SEQ ID NO: 14, FR1 is defined by amino acid residues 1-26, FR2 is defined by amino acid residues 33-49, FR3 is defined by amino acid residues 53-88, and FR4 is defined by amino acid residues 98-107.

[0085] The present disclosure further provides a multispecific antibody, wherein the VH sequence of the FAPα-binding region comprises or consists of the VH sequence set forth in SEQ ID NO: 13. The present disclosure further provides a multispecific antibody, wherein the VL sequence of the FAPα-binding region comprises or consists of the VH sequence set forth in SEQ ID NO: 14. In a further embodiment, the VH and VL sequences of the FAPα-binding region comprise or consist of the VH sequence set forth in SEQ ID NO: 13 and the VL sequence set forth in SEQ ID NO: 14. In a further embodiment, the VH sequence of the FAPα-binding region comprises, consists essentially of, or consists of the VH sequence set forth in SEQ ID NO: 13. In yet a further embodiment, the VL sequence of the FAPα-binding region comprises, consists essentially of, or consists of the VL sequence set forth in SEQ ID NO: 14. In yet a further embodiment, the VH and VL sequences of the FAPα-binding region comprise, consist essentially of, or consist of the VH sequence set forth in SEQ ID NO: 13 and the VL sequence set forth in SEQ ID NO: 14.

[0086] An antibody in the context of the present invention may comprise a FAPα-binding region capable of binding to FAPα, wherein FAPα is human FAPα, for example, the mature polypeptide of SEQ ID NO: 33 or the soluble FAPα of SEQ ID NO: 34; FAPα is mouse FAPα, for example, the mature polypeptide of SEQ ID NO: 35; FAPα is rat FAPα, for example, the mature polypeptide of SEQ ID NO: 36; FAPα is canine FAPα, for example, the mature polypeptide of SEQ ID NO: 37; FAPα is porcine FAPα, for example, the mature polypeptide of SEQ ID NO: 38, or cynomolgus FAPα, for example, the mature polypeptide of SEQ ID NO: 39. In a further embodiment, the FAPα is human FAPα, for example, the mature polypeptide of SEQ ID NO: 33 or the soluble FAPα of SEQ ID NO: 34, or cynomolgus FAPα, for example, the mature polypeptide of SEQ ID NO: 39. In a preferred embodiment, the FAPα is human FAPα, for example, the mature polypeptide of SEQ ID NO: 33.

[0087] The antibody according to the present invention has an equilibrium dissociation constant K between the antigen-binding region that binds to human FAPα and human FAPα.D can be bonded with K D is 1000pM or less, for example 900pM or less, for example 800pM or less, for example 700pM or less, for example 600pM or less, for example 500pM or less, for example 400pM or less, for example 300pM or less, for example 200pM or less, for example 100pM or less, for example 90pM or less, for example 80pM or less, for example 70pM or less, for example 60pM or less, for example 50pM or less, for example 40pM or less, for example 3 The binding affinity is 0 pM or less, or is within the range of 0.1 pM to 1000 pM, for example, 0.5-900 pM, for example, 1 pM to 800 pM, for example, 2 pM to 700 pM, for example, 3 pM to 600 pM, for example, 4 pM to 500 pM, for example, 5 pM to 400 pM, for example, 6 pM to 300 pM, for example, 7 pM to 200 pM, for example, 8 pM to 100 pM, for example, 9 pM to 75 pM, for example, 10 pM to 50 pM. The binding affinity can be determined by biolayer interferometry.

[0088] In a further embodiment, the equilibrium dissociation constant K D When binding monovalently, is 1000 pM or less, for example 900 pM or less, for example 800 pM or less, for example 700 pM or less, for example 600 pM or less, for example 500 pM or less, for example 400 pM or less, for example 300 pM or less, for example 200 pM or less, for example 100 pM or less, for example 90 pM or less, for example 80 pM or less, for example 70 pM or less, for example 60 pM or less, for example 50 pM or less, for example 40 pM or less , for example, 30 pM or less, or within the range of 0.1 pM to 1000 pM, for example, 0.5-900 pM, for example, 1 pM to 800 pM, for example, 2 pM to 700 pM, for example, 3 pM to 600 pM, for example, 4 pM to 500 pM, for example, 5 pM to 400 pM, for example, 6 pM to 300 pM, for example, 7 pM to 200 pM, for example, 8 pM to 100 pM, for example, 9 pM to 75 pM, for example, 10 pM to 50 pM. Binding affinity can be determined by biolayer interferometry.

[0089] Multispecific antibodies that bind to human lung fibroblasts or CAFs have an EC 200 for FAPα binding, when assayed, for example, as described in Example 2 herein. 50 Further disclosed herein are multispecific antibodies, wherein the total antibody titer is in the range of 0.01 to 0.5 μg / mL, for example, in the range of 0.02 to 0.4 μg / mL, for example, in the range of 0.03 to 0.3 μg / mL.

[0090] When the multispecific antibody binds monovalently to human lung fibroblasts, it has an EC 200 for FAPα binding, when assayed, for example, as described in Example 2 herein. 50 Further disclosed herein are multispecific antibodies, wherein the total antibody concentration is in the range of 0.01 to 0.5 μg / mL, for example, in the range of 0.02 to 0.1 μg / mL, for example, in the range of 0.03 to 0.5 μg / mL.

[0091] When the multispecific antibody binds bivalently to human lung fibroblasts, it has an EC 200 for FAPα binding, when assayed, for example, as described in Example 2 herein. 50 Further disclosed herein are multispecific antibodies, wherein the total antibody titer is in the range of 0.01 to 0.05 μg / mL, for example, in the range of 0.01 to 0.03 μg / mL, for example, in the range of 0.01 to 0.02 μg / mL.

[0092] Multispecific antibodies that bind monovalently to CAFs have an EC of 1.2 for FAPα binding, when assayed, for example, as described in Example 2 herein. 50 Further disclosed herein is a multispecific antibody having a ribonucleotide concentration in the range of 0.1 to 0.5 μg / mL, for example, in the range of 0.15 to 0.4 μg / mL, for example, in the range of 0.2 to 0.3 μg / mL. The DR4-binding region of the multispecific antibody may comprise a heavy chain variable region (VH) comprising three complementarity determining regions, CDR1, CDR2, and CDR3, present within the amino acid sequence set forth in SEQ ID NO: 15.

[0093] The DR4 binding region of the multispecific antibody may comprise a light chain variable region (VL) comprising the three complementarity determining regions, CDR1, CDR2 and CDR3, present in the amino acid sequence set forth in SEQ ID NO: 16. In one embodiment, the DR4 binding region of the multispecific antibody comprises a heavy chain variable region (VH) comprising the three complementarity determining regions, CDR1, CDR2 and CDR3, present in the amino acid sequence set forth in SEQ ID NO: 15, and a light chain variable region (VL) comprising the three complementarity determining regions, CDR1, CDR2 and CDR3, present in the amino acid sequence set forth in SEQ ID NO: 16.

[0094] Further disclosed herein is a multispecific antibody, wherein the DR4 binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 8 and 9, respectively. Also disclosed herein is a multispecific antibody, wherein the DR4 binding region comprises a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, 11 and 12, respectively. In a further embodiment, the DR4 binding region of the multispecific antibody comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 8 and 9, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, 11 and 12, respectively. The CDR regions from the variable heavy and light chain regions are annotated according to IMGT (see Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, Developmental and Comparative Immunology, 27(1), 55-77 (2003)).

[0095] The present disclosure further provides multispecific antibodies wherein the VH sequence of the DR4 binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. The present disclosure also provides multispecific antibodies wherein the VL sequence of the DR4 binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 16. In further embodiments, the VH sequence of the DR4 binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 15, and said VL sequence of the DR4 binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 16.

[0096] Furthermore, the multispecific antibody may further comprise a framework region of the VH sequence of the DR4 binding region that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. Furthermore, the multispecific antibody may further comprise a framework region of the VL sequence of the DR4 binding region that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 16. In further embodiments, the framework regions of the VH sequence of the DR4 binding domain have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 15, and said framework regions of the VL sequence of the DR4 binding domain have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 16.

[0097] In further embodiments, the framework regions of the VH sequence of the DR4 binding region of the multispecific antibody described herein have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the framework regions of the amino acid sequence set forth in SEQ ID NO: 15. In still further embodiments, the framework regions of the VL sequence of the DR4 binding region described herein have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the framework regions of the amino acid sequence set forth in SEQ ID NO: 16. In still further embodiments, the framework regions of the VH sequence of the DR4 binding region have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the framework regions of the amino acid sequence set forth in SEQ ID NO: 15, and said framework regions of the VL sequence of the DR4 binding region have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the framework regions of the amino acid sequence set forth in SEQ ID NO: 16.

[0098] In a further embodiment, said VH sequence of the DR4 binding region as set forth in SEQ ID NO: 15 comprises up to 10 substitutions, such as up to 9 substitutions, for example up to 8 substitutions, such as up to 7 substitutions, for example up to 6 substitutions, such as up to 5 substitutions, for example up to 4 substitutions, such as up to 3 substitutions, for example up to 2 substitutions, such as up to 1 substitution. In a still further embodiment, said VL sequence of the DR4 binding region as set forth in SEQ ID NO: 16 comprises up to 10 substitutions, such as up to 9 substitutions, for example up to 8 substitutions, such as up to 7 substitutions, for example up to 6 substitutions, such as up to 5 substitutions, for example up to 4 substitutions, for example up to 3 substitutions, such as up to 2 substitutions, for example up to 1 substitution. In a further embodiment, said VH sequence of the DR4 binding region differs from SEQ ID NO: 15 by up to 10 substitutions, such as up to 9 substitutions, for example up to 8 substitutions, such as up to 7 substitutions, for example up to 6 substitutions, such as up to 5 substitutions, for example up to 4 substitutions, for example up to 3 substitutions, such as up to 2 substitutions, for example up to 1 substitution. In yet a further embodiment, said VL sequence of the DR4 binding region differs from SEQ ID NO: 16 by up to 10 substitutions, such as up to 9 substitutions, for example up to 8 substitutions, such as up to 7 substitutions, for example up to 6 substitutions, such as up to 5 substitutions, for example up to 4 substitutions, such as up to 3 substitutions, for example up to 2 substitutions, such as up to 1 substitution.

[0099] In yet a further embodiment, the VH and VL sequences of the DR4 binding domain differ only in the framework regions. In one embodiment, in the VH sequence according to SEQ ID NO: 15, FR1 is defined by amino acid residues 1 to 25, FR2 is defined by amino acid residues 35 to 51, FR3 is defined by amino acid residues 59 to 96, and FR4 is defined by amino acid residues 106 to 116. In another embodiment, in the VL sequence according to SEQ ID NO: 16, FR1 is defined by amino acid residues 1 to 25, FR2 is defined by amino acid residues 35 to 51, FR3 is defined by amino acid residues 55 to 90, and FR4 is defined by amino acid residues 101 to 110.

[0100] The present disclosure further provides a multispecific antibody, wherein the VH sequence of the DR4 binding region comprises or consists of the VH sequence set forth in SEQ ID NO: 15. The present disclosure further provides a multispecific antibody, wherein the VL sequence of the DR4 binding region comprises or consists of the VL sequence set forth in SEQ ID NO: 16. In a further embodiment, the VH and VL sequences of the DR4 binding region comprise or consist of the VH sequence set forth in SEQ ID NO: 15 and the VL sequence set forth in SEQ ID NO: 16. In a further embodiment, the VH sequence of the DR4 binding region comprises, consists essentially of, or consists of the VH sequence set forth in SEQ ID NO: 15. In yet a further embodiment, the VL sequence of the DR4 binding region comprises, consists essentially of, or consists of the VL sequence set forth in SEQ ID NO: 16. In yet a further embodiment, the VH and VL sequences of the DR4 binding region comprise, consist essentially of, or consist of the VH sequence set forth in SEQ ID NO: 15 and the VL sequence set forth in SEQ ID NO: 16.

[0101] An antibody in the context of the present invention may comprise a DR4 binding region capable of binding to DR4, wherein DR4 is human DR4, such as the mature polypeptide of SEQ ID NO: 68, or cynomolgus DR4, such as the mature polypeptide of SEQ ID NO: 69. In a further embodiment, the DR4 is human DR4, such as the mature polypeptide of SEQ ID NO: 68.

[0102] The antibody according to the present invention has an equilibrium dissociation constant K between the antigen-binding region that binds to human DR4 and human DR4. D can be bonded with K Dis 100nM or less, for example 90nM or less, for example 80nM or less, for example 70nM or less, for example 60nM or less, for example 50nM or less, for example 40nM or less, for example 30nM or less, for example 20nM or less, for example 10nM or less, for example 9nM or less, for example 8nM or less, for example 7nM or less, for example 6nM or less, for example 5nM or less, for example 4nM or less, for example 3nM or less, for example 2nM or less, for example 1nM or less, e.g. For example, 0.5 nM or less, or within the range of 0.01 nM to 10 nM, for example, 0.02 nM to 9 nM, for example, 0.03 nM to 8 nM, for example, 0.04 nM to 7 nM, for example, 0.05 nM to 6 nM, for example, 0.075 nM to 5 nM, for example, 0.1 nM to 4 nM, for example, 0.15 nM to 3 nM, for example, 0.2 nM to 2 nM, for example, 0.25 nM to 1 nM, for example, 0.3 nM to 0.75 nM (monovalent binding). Binding affinity can be determined by biolayer interferometry.

[0103] In a further embodiment, the equilibrium dissociation constant K of the antigen-binding region that binds to human DR4 is D When binding monovalently, it is 100 nM or less, for example 90 nM or less, for example 80 nM or less, for example 70 nM or less, for example 60 nM or less, for example 50 nM or less, for example 40 nM or less, for example 30 nM or less, for example 20 nM or less, for example 10 nM or less, for example 9 nM or less, for example 8 nM or less, for example 7 nM or less, for example 6 nM or less, for example 5 nM or less, for example 4 nM or less, for example 3 nM or less, for example 2 nM or less, for example For example, the binding affinity may be 1 nM or less, for example, 0.5 nM or less, or within the range of 0.01 nM to 10 nM, for example, 0.02 nM-9 nM, for example, 0.03 nM-8 nM, for example, 0.04 nM to 7 nM, for example, 0.05 nM to 6 nM, for example, 0.075 nM to 5 nM, for example, 0.1 nM to 4 nM, for example, 0.15 nM to 3 nM, for example, 0.2 nM to 2 nM, for example, 0.25 nM to 1 nM, for example, 0.3 nM to 0.75 nM. The binding affinity may be determined by biolayer interferometry.

[0104] Multispecific antibodies that bind to DLD-1, A549, HCT-116, HCT-15, MDA-MB-231, or PANC-1 have an EC 20 value for binding to DR4, e.g., when assayed as described in Example 3 herein. 50 Further disclosed herein are multispecific antibodies, wherein the total antibody titer is in the range of 0.1 to 3.0 μg / mL, for example, in the range of 0.2 to 2.5 μg / mL, for example, in the range of 0.3 to 2.0 μg / mL.

[0105] Multispecific antibodies that monovalently bind to DLD-1, A549, HCT-116, HCT-15, MDA-MB-231, or PANC-1 have an EC 20 or EC 20 for binding to DR4, when assayed, for example, as described in Example 3 herein. 50 Further disclosed herein are multispecific antibodies, wherein the IgG antibody concentration is in the range of 0.1 to 3.0 μg / mL, for example, in the range of 0.25 to 2.0 μg / mL, for example, in the range of 0.3 to 1.75 μg / mL.

[0106] Multispecific antibodies that bivalently bind DLD-1, A549, HCT-116, HCT-15, MDA-MB-231, or PANC-1 have an EC 20 or EC 20 binding to DR4, when assayed, for example, as described in Example 3 herein. 50 Further disclosed herein are multispecific antibodies, wherein the IgG antibody concentration is in the range of 0.1 to 1.0 μg / mL, for example, in the range of 0.1 to 0.75 μg / mL, for example, in the range of 0.1 to 0.5 μg / mL.

[0107] The present disclosure further provides a multispecific antibody that is a bispecific antibody. In one embodiment, the antibody comprises (i) a FAPα-binding region comprising a first heavy chain variable region and a first light chain variable region, wherein the heavy chain variable region (VH) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 1, 2, and 3, respectively, and the light chain variable region (VL) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 4, 5, and 6, respectively, and (ii) a DR4-binding region comprising a second heavy chain variable region and a second light chain variable region, wherein the heavy chain variable region (VH) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 7, 8, and 9, respectively, and the light chain variable region (VL) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 10, 11, and 12, respectively. In a further embodiment, the antibody comprises (i) a FAPα-binding region comprising, or consisting of, the VH sequence set forth in SEQ ID NO: 13 and the VL sequence set forth in SEQ ID NO: 14, and (ii) a DR4-binding region comprising, or consisting of, the VH sequence set forth in SEQ ID NO: 15 and the VL sequence set forth in SEQ ID NO: 16. In another embodiment, the bispecific antibody comprises (i) a FAPα-binding region comprising, consisting essentially of, or consisting of the VH sequence set forth in SEQ ID NO: 13 and the VL sequence set forth in SEQ ID NO: 14, and (ii) a DR4-binding region comprising, consisting essentially of, or consisting of the VH sequence set forth in SEQ ID NO: 15 and the VL sequence set forth in SEQ ID NO: 16.

[0108] The antibody according to the present invention has an equilibrium dissociation constant K between the antigen-binding region that binds to human FAPα and human FAPα of 100 pM or less. D and the equilibrium dissociation constant K between the antigen-binding region that binds to human DR4 and human DR4 is 1 nM or less. D The binding affinity can be determined by biolayer interferometry.

[0109] The antibody according to the present invention has an equilibrium dissociation constant K between the antigen-binding region that binds to human FAPα and human FAPα of 100 pM or less. DThe antigen-binding region can bind to human DR4 via a monovalent bond (monovalent binding), and the equilibrium dissociation constant K between the antigen-binding region that binds to human DR4 and human DR4 is 1 nM or less. D (monovalent binding). The binding affinity can be determined by biolayer interferometry.

[0110] The antibody according to the present invention has an equilibrium dissociation constant K between the antigen-binding region that binds to human FAPα and human FAPα of 50 pM or less. D The antigen-binding region can bind to human DR4 via a monovalent bond (monovalent binding), and the equilibrium dissociation constant K between the antigen-binding region that binds to human DR4 and human DR4 is 0.5 nM or less. D (monovalent binding). The binding affinity can be determined by biolayer interferometry.

[0111] In a further aspect, the present invention relates to a multispecific antibody comprising at least one antigen-binding region capable of binding to FAPα, wherein the antibody is capable of competing for binding to FAPα with the antibody FAP-ESC11, e.g. as disclosed in WO2011040972, comprising a heavy chain (HC) comprising the sequence set forth in SEQ ID NO: 50 and a light chain (LC) comprising the sequence set forth in SEQ ID NO: 51, and / or is capable of simultaneously binding to FAPα as the antibody FAP5, e.g. as disclosed in US Patent Application Publication No. 20090304718, comprising a heavy chain (HC) comprising the sequence set forth in SEQ ID NO: 48 and a light chain (LC) comprising the sequence set forth in SEQ ID NO: 49.

[0112] Antibody Format The multispecific antibody of the present invention may have two or more specificities, for example, two or three or more specificities. Furthermore, the multispecific antibody may have multiple copies of the antigen-binding region for FAPα and / or DR4. For example, in one embodiment, the antibody has two antigen-binding regions capable of binding to FAPα, for example, two identical binding regions that bind to FAPα. For example, in another embodiment, the antibody has two antigen-binding regions that bind to DR4, for example, two identical binding regions that bind to DR4. The additional antigen-binding region may be present, for example, in the form of an scFv covalently linked to the constant region.

[0113] In a preferred embodiment, the multispecific antibody of the present invention is a bispecific antibody.Various formats and uses of bispecific antibodies are known in the art and have been reviewed by Kontermann; Drug Discov Today, 2015 Jul; 20(7): 838-47, and MAbs, 2012 Mar-Apr; 4(2): 182-97, and by Labrijn et al. 2019 Nat Rev Drug Discov 18(8) 585-608.The bispecific antibody of the present invention is not limited to a particular bispecific format or its production method.

[0114] Examples of bispecific antibody molecules that can be used in the present invention include: (i) a single antibody with two arms containing different antigen-binding regions; (ii) a single-chain antibody with specificity for two different targets, e.g., via two scFvs linked in tandem by an additional peptide linker; and (iii) a dual variable domain antibody (DVD-Ig), in which the light and heavy chains each contain two variable regions in tandem via a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin. Heidelberg (2010)), (iv) chemically linked bispecific (Fab') fragments, (v) Tandabs, which are fusions of two single-chain diabodies resulting in tetravalent bispecific antibodies with two binding sites for each target antigen, (vi) Flexibodies, which are combinations of scFvs and diabodies resulting in multivalent molecules, (vii) so-called "dock and lock" molecules based on the "dimerization and docking domain" of protein kinase A, which when applied to Fabs can result in trivalent bispecific binding proteins consisting of two identical Fab fragments linked to different Fab fragments, (viii) so-called Scorpion molecules, which contain, for example, two scFvs fused to either end of a human Fab arm, and (ix) diabodies.

[0115] Further examples of different classes of bispecific antibodies include, but are not limited to, (i) IgG-like molecules that have complementary CH3 domains and force heterodimerization; (ii) recombinant IgG-like dual targeting molecules, in which each side of the molecule contains an Fab fragment or portion of an Fab fragment of at least two different antibodies; (iii) IgG fusion molecules, in which a full-length IgG antibody is fused to an additional Fab fragment or portion of an Fab fragment; (iv) Fc fusion molecules, in which a single-chain Fv molecule or stabilized diabody is fused to a heavy-chain constant domain, Fc region or portion thereof; (v) Fab fusion molecules, in which different Fab fragments are fused together and fused to a heavy-chain constant domain, Fc region or portion thereof; and (vi) scFv- and diabody-based and heavy-chain antibodies (e.g., domain antibodies, nanobodies) in which different single-chain Fv molecules or different diabodies or different heavy-chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule fused to a heavy-chain constant domain, Fc region or portion thereof.

[0116] Examples of IgG-like molecules with complementary CH3 domain molecules include, but are not limited to, Triomab / Quadroma molecules (Trion Pharma / Fresenius Biotech; Roche, WO 2011069104), so-called Knobs-into-Holes molecules (Genentech, WO 9850431), CrossMAb (Roche, WO 2011117329) and electrostatically-matched molecules (Amgen, EP 1870459 and WO 2009089004; Chugai, U.S. Patent Application Publication No. 201000155133; Oncomed, WO 2010129304), LUZ-Y molecules (Genentech, Wranik et al., 20100129304), and the like. al. J. Biol. Chem. 2012, 287(52):43331-9, doi:10.1074 / jbc.M112.397869. Epub 2012 Nov 1), DIG body and PIG body molecules (Pharmabcine, WO 2010134666, WO 2014081202), Strand Exchange Engineered Domain body (SEEDbody) molecules (EMD Serono, WO 2007110205), Biclonics molecules (Merus, WO 2013157953), FcΔAdp molecules (Regeneron, WO 201015792), bispecific IgG1 and IgG2 molecules (Pfizer / Rinat, WO 11143545), Azymetric scaffold molecules (Zymeworks / Merck, WO 2012058768), mAb-Fv molecules (Xencor, WO 2011028952), bivalent bispecific antibodies (WO 2009080254) and DuoBody® molecules (Genmab A / S, WO 2011131746).

[0117] Examples of recombinant IgG-like dual targeting molecules include, but are not limited to, Dual Targeting (DT)-Ig molecules (WO 2009058383), Two-in-one antibodies (Genentech; Bostrom, et al, 2009. Science 323, 1610-1614), cross-linked Mab (Karmanos Cancer Center), mAb2 (F-Star, WO 2008003116), Zybody molecules (Zyngenia; LaFleur et al. MAbs. 2013 Mar-Apr; 5(2):208-18), common light chain approaches (Crucell / Merus, U.S. Patent No. 7,262,028), kappa / lambda body™ molecules (NovImmune, WO 2012023053), and CovX bodies (CovX / Pfizer; Doppalapudi, VR, et al. 2007. Bioorg. Med. Chem. Lett. 17, 501-506).

[0118] Examples of IgG fusion molecules include, but are not limited to, dual variable domain (DVD)-Ig molecules (Abbott, U.S. Pat. No. 7,612,181), dual-domain double-headed antibodies (Unilever; Sanofi Aventis, WO 20100226923), IgG-like bispecific molecules (ImClone / Eli Lilly, Lewis et al. Nat Biotechnol. 2014 Feb;32(2):191-8), Ts2Ab (MedImmune / AZ; Dimasi et al. J Mol Biol. 2009 Oct 30;393(3):672-92) and BsAb molecules (Zymogenetics, WO 2010111625), HERCULES molecules (Biogen Idec, U.S. Pat. No. 007951918), scFv fusion molecules (Novartis), scFv fusion molecules (Changzhou Adam Biotech Inc., China Patent No. 102250246) and TvAb molecules (Roche, WO 2012025525, WO 2012025530).

[0119] Examples of Fc fusion molecules include, but are not limited to, scFv / Fc fusions (Pearce et al., Biochem Mol Biol Int. 1997 Sep;42(6):1179-88), SCORPION molecules (Emergent BioSolutions / Trubion, Blankenship JW, et al. AACR 100th Annual Meeting 2009 (Abstract #5465); Zymogenetics / BMS, WO 2010111625), Dual Affinity Retargeting Technology (Fc-based DART) molecules (MacroGenics, WO 2008157379, WO 2010080538), and Dual(scFv)2-Fab molecules (National Research Center for Antibody Medicine-China).

[0120] Examples of Fab-fused bispecific antibodies include, but are not limited to, F(ab)2 molecules (Medarex / AMGEN; Deo et al J Immunol. 1998 Feb 15;160(4):1677-86), dual-acting or Bis-Fab molecules (Genentech, Bostrom, et al 2009. Science 323,1610-1614), Dock-and-Lock (DNL) molecules (ImmunoMedics, WO 2003074569, WO 2005004809), bivalent bispecific molecules (Biotechnol, Schoonjans, J Immunol. 2000 Dec 15;165(12):7050-7), and Fab-Fv molecules (UCB-Celltech, WO 2009040562 A1).

[0121] Examples of scFv-based, diabody-based, and domain antibodies include, but are not limited to, bispecific T cell engager (BiTE) molecules (Micromet, WO 2005061547), tandem diabody molecules (TandAb) (Affimed) (Le Gall et al., Protein Eng Des Sel. 2004 Apr;17(4):357-66.), DART molecules (MacroGenics, WO 2008157379, WO 2010080538), single-chain diabody molecules (Lawrence, FEBS Lett. 1998 Apr 3;425(3):479-84), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin scFv fusions (Merrimack, WO 2010059315), and COMBODY molecules (Epigen Biotech, Zhu et al. al. Immunol Cell Biol. 2010 Aug;88(6):667-75.), dual-targeting nanobodies (Ablynx, Hmila et al., FASEB J. 2010) and dual-targeting heavy chain single domain antibodies.

[0122] In one embodiment, the bispecific antibody of the invention is a diabody, a crossbody, or a bispecific antibody obtained via controlled Fab arm exchange (e.g., as described in WO2011131746 (Genmab)).

[0123] In one embodiment, an antibody of the invention is a bispecific DuoBody® molecule (Genmab A / S, WO 2011131746).

[0124] The multispecific, e.g., bispecific, antibodies of the present invention can be of any isotype. Exemplary isotypes include, but are not limited to, any of the human IgG1, IgG2, IgG3, and IgG4 isotypes. Preferably, the antibodies can be selected to be of the human IgG1 isotype, as shown in the Examples. Thus, in one embodiment, the multispecific antibody is an IgG1 antibody. Human light chain constant regions, either kappa or lambda, or both, can be used, e.g., the sequences set forth in SEQ ID NOs: 27 and 28. In one embodiment, the multispecific antibody comprises kappa (κ) and lambda (λ) light chains. For example, in one embodiment, the light chain involved in FAPα binding comprises a kappa constant region, and the light chain involved in DR4 binding comprises a lambda constant region. In a further embodiment, the multispecific antibody comprises a heavy chain and a kappa (κ) light chain comprising a FAPα-binding region, and a heavy chain and a lambda (λ) light chain comprising a DR4-binding region. In one embodiment, both heavy chains of the antibody of the present invention are of the IgG1 isotype. In a further embodiment, the two heavy chains of the bispecific antibody are of the IgG1 and IgG4 isotypes, respectively. In yet a further embodiment, the DR4 binding region is comprised in a heavy chain and a light chain, the heavy chain comprising the VH region and an IgG1 heavy chain constant region, the light chain comprising the VL region and a lambda light chain constant region, the FAPα binding region is comprised in a heavy chain and a light chain, the heavy chain comprising the VH region and an IgG1 heavy chain constant region, and the light chain comprising the VL region and a kappa light chain constant region. In yet a further embodiment, one IgG1 heavy chain constant region is as defined in SEQ ID NO:26 and the other is as defined in SEQ ID NO:70, the kappa light chain constant region is as defined in SEQ ID NO:27, and the lambda light chain constant region is as defined in SEQ ID NO:28.

[0125] Preferably, the bispecific antibody may be selected to be of the human IgG1 isotype, as shown in the examples. Optionally, and preferably, the heavy chain and its Fc region sequence of the selected isotype may be modified, preferably in the hinge, CH2 and / or CH3 regions, to allow the generation of bispecific antibodies and / or to introduce inactivation.

[0126] In one embodiment, a multispecific antibody of the invention comprises an Fc region consisting of a first and a second Fc polypeptide.

[0127] In one embodiment, the first Fc polypeptide and the first heavy chain variable region are comprised within the same polypeptide chain, and the second Fc polypeptide and the second heavy chain variable region are comprised within the same polypeptide chain.

[0128] The first and second Fc polypeptides can each be of any isotype, including any human isotype, such as IgG1, IgG2, IgG3, IgG4, IgE, IgD, IgM, or IgA, or mixed isotypes. Preferably, the Fc region is a human IgG1, IgG2, IgG3, IgG4 isotype, or mixed isotypes. In one embodiment, the Fc region is a human IgG1 Fc region.

[0129] In a further embodiment, the multispecific antibody is a full-length antibody as defined herein. In yet a further embodiment, the multispecific antibody is a full-length IgG1 antibody. In yet a further embodiment, the first and second Fc regions comprise the sequence of SEQ ID NO: 21 (IgG1m(f)), except for the specific mutations defined herein.

[0130] The antibodies of the present invention may contain modifications in the Fc region to render them inactive or non-activating. Thus, in the antibodies disclosed herein, one or both heavy chains may be modified to reduce the extent of Fc-mediated effector function induced by the antibody compared to an otherwise identical antibody lacking the modifications. Thus, in one embodiment, the antibody comprises a first heavy chain and a second heavy chain, and one or both heavy chains are modified to reduce the extent of Fc-mediated effector function induced by the antibody compared to an otherwise identical antibody comprising unmodified first and second heavy chains. Fc-mediated effector function can be measured by binding to Fcγ receptors, by binding to C1q, or by induction of Fc-mediated cross-linking of FcγRs. In particular, modifications of the heavy and light chain constant sequences may also result in reduced C1q binding to the antibody. The reduction may be at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to an unmodified antibody, and C1q binding may be determined by ELISA. Furthermore, the Fc region may be modified to reduce Fc-mediated T-cell proliferation mediated by said antibody by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100%, e.g., in the linear portion of the curve, compared to an unmodified antibody, wherein said T-cell proliferation is measured in a PBMC-based functional assay. In one embodiment, the multispecific antibody induces Fc-mediated effector function to less than 95%, such as less than 90%, less than 85%, such as less than 80%, for example less than 75%, for example less than 70%, for example less than 65%, for example less than 60%, for example less than 55%, for example less than 50% of an identical antibody except for comprising unmodified first and second heavy chains.

[0131] A wide variety of non-activated antibody formats have been developed in which amino acid substitutions and combinations thereof have been introduced into the constant heavy chain region of IgG1 isotype antibodies to eliminate Fc-mediated effector functions (e.g., Chiu et al., Antibodies 2019 Dec;8(4):55; Liu et al., Antibodies, 2020 Nov 17;9(4):64;29(10):457-66; Shields et al., J Biol Chem. 2001 Mar 2;276(9):6591-604). In one embodiment, a multispecific antibody comprises a first heavy chain and a second heavy chain, wherein in at least one of the first heavy chain and the second heavy chain, one or more amino acids at positions corresponding to positions L234, L235, G236, D265, N297, and P331 of a human IgG1 heavy chain according to EU numbering are not L, L, G, D, N, or P, respectively.

[0132] For example, examples of amino acid positions that may be modified in an IgG1 isotype antibody include positions L234 and L235. Thus, in one embodiment, the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to Eu numbering are F and E, respectively, in the first and / or second heavy chain.

[0133] It is understood that in addition to modification of amino acid positions L234 and L235, additional positions may be modified. Thus, in a further embodiment, the first and second Fc polypeptides comprise substitutions of amino acids corresponding to positions L234 and L235 with F and E, respectively, and the first and / or second Fc polypeptides further comprise substitutions of amino acids corresponding to position G236 of a human IgG1 heavy chain, preferably with R.

[0134] In another embodiment, the first and second Fc polypeptides comprise substitutions of amino acids corresponding to amino acids at positions L234 and L235 with F and E, respectively, and the first and second Fc polypeptides further comprise substitutions of amino acids corresponding to amino acid at position G236 of a human IgG1 heavy chain, preferably with R.

[0135] In another embodiment, the first and second Fc polypeptides comprise substitutions of amino acids corresponding to amino acids at positions L234 and L235 with F and E, respectively, and the first and / or second Fc polypeptides further comprise substitutions of amino acids corresponding to amino acids at position D265 of a human IgG1 heavy chain, preferably with A.

[0136] In another embodiment, the first and second Fc polypeptides comprise substitutions of amino acids corresponding to amino acids at positions L234 and L235 with F and E, respectively, and the first and second Fc polypeptides further comprise substitutions of amino acids corresponding to amino acids at position D265 of a human IgG1 heavy chain, preferably with A.

[0137] In another embodiment, one of the first Fc polypeptide and the second Fc polypeptide comprises substitutions of amino acids corresponding to amino acids at positions L234, L235, and G236 with F, E, and R, respectively, and the other Fc polypeptide comprises substitutions of amino acids corresponding to amino acids at positions L234, L235E, and D265 with F, E, and A, respectively.

[0138] For example, constant regions with such Fc region substitutions are provided in SEQ ID NOs: 22-23, which can be compared to SEQ ID NO: 21 without such substitutions. In one embodiment, an antibody of the invention comprises a sequence selected from the group consisting of SEQ ID NOs: 22-23.

[0139] In one embodiment, the multispecific or bispecific antibody of the present invention comprises an Fc region comprising different first and second CH3 regions, and the heterodimeric interaction between the first and second CH3 regions is stronger than the homodimeric interaction between the first and second CH3 regions, respectively. Further details about these interactions and how they can be achieved are provided in WO2011131746 and WO2013060867 (Genmab), which are incorporated herein by reference. Stable heterodimeric antibodies can be obtained in high yields based on two homodimeric starting antibodies containing only minor asymmetric mutations in the CH3 regions, for example, by so-called Fab arm exchange, as provided in WO2008 / 119353 and WO2011 / 131746.

[0140] Thus, in one embodiment, a first Fc polypeptide comprises a substitution of at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 of a human IgG1 heavy chain, and a second Fc polypeptide comprises a substitution of at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 of a human IgG1 heavy chain, wherein the substitutions in the first and second Fc polypeptides are not at the same positions, and the amino acid positions are as defined by EU numbering. For example, constant regions having such Fc region substitutions are provided in SEQ ID NOs: 24-25, which can be compared to SEQ ID NO: 21, which does not have such substitutions. In one embodiment, an antibody of the invention comprises a sequence selected from the group consisting of SEQ ID NOs: 24-25.

[0141] Further disclosed herein is a multispecific antibody, (i) wherein the antibody comprises a first heavy chain and a second heavy chain, wherein the first heavy chain comprises the FAPα-binding region and the second heavy chain comprises the DR4-binding region; (ii) wherein the first heavy chain and the second heavy chain each comprise at least a hinge region, a CH2 region, and a CH3 region; and (iii) wherein the first heavy chain has at least one amino acid substitution at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (according to EU numbering), and the second heavy chain has at least one amino acid substitution at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (according to EU numbering).

[0142] Further disclosed herein is a multispecific antibody, (i) wherein the antibody comprises a first heavy chain and a second heavy chain, wherein said first heavy chain comprises the VH region of a FAPα-binding region and said second heavy chain comprises the VH region of a DR4-binding region; (ii) wherein said first heavy chain and said second heavy chain each comprise at least a hinge region, a CH2 and a CH3 region; and (iii) wherein in said first heavy chain, and in said second heavy chain at least one amino acid substitution is made at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 (in a human IgG1 heavy chain according to Eu numbering), and

[0143] Further disclosed herein are multispecific antibodies, wherein the first heavy chain and the second heavy chain are not substituted at the same positions. Further disclosed are multispecific antibodies, wherein (i) the amino acid at the position corresponding to F405 (according to Eu numbering) is L in the first heavy chain and the amino acid at the position corresponding to K409 (according to Eu numbering) is R in the second heavy chain, or (ii) the amino acid at the position corresponding to K409 (according to Eu numbering) is R in the first heavy chain and the amino acid at the position corresponding to F405 (according to Eu numbering) is L in the second heavy chain. Preferably, the amino acid at the position corresponding to F405 is L in the first Fc polypeptide and the amino acid at the position corresponding to K409 is R in the second Fc polypeptide, or vice versa. Thus, the present invention provides antibodies in which the amino acid at the position corresponding to F405 of the human IgG1 heavy chain is L in the first Fc polypeptide and the amino acid at the position corresponding to K409 of the human IgG1 heavy chain is R in the second Fc polypeptide, or vice versa. In further embodiments, multispecific antibodies are disclosed, in which (i) the amino acid at the position corresponding to F405 (in the human IgG1 heavy chain according to Eu numbering) is L in the first heavy chain and the amino acid at the position corresponding to K409 (in the human IgG1 heavy chain according to Eu numbering) is R in the second heavy chain, or (ii) the amino acid at the position corresponding to K409 (in the human IgG1 heavy chain according to Eu numbering) is R in the first heavy chain and the amino acid at the position corresponding to F405 (in the human IgG1 heavy chain according to Eu numbering) is L in the second heavy chain.

[0144] Thus, in one embodiment, one of the first and second Fc polypeptides comprises substitutions of amino acids corresponding to positions L234, L235, G236 and F405 with F, E, R and L, respectively, and the other Fc polypeptide comprises substitutions of amino acids corresponding to positions L234, L235E, D265 and K409 with F, E, A and R, respectively.

[0145] In another embodiment, one of the first Fc polypeptide and the second Fc polypeptide comprises substitutions of amino acids corresponding to amino acids at positions L234, L235, G236 and K409 with F, E, R and R, respectively, and the other Fc polypeptide comprises substitutions of amino acids corresponding to amino acids at positions L234, L235E, D265 and F405 with F, E, A and L, respectively.

[0146] In another embodiment, one of the first Fc polypeptide and the second Fc polypeptide comprises substitutions of amino acids corresponding to amino acids at positions L234, L235, G236 and F405 with F, E, R and L, respectively, and the other Fc polypeptide comprises substitutions of amino acids corresponding to amino acids at positions L234, L235E, G236 and K409 with F, E, R and R, respectively.

[0147] In a further embodiment, the first Fc polypeptide comprises substitutions of amino acids corresponding to amino acids at positions L234, L235, G236 and F405 with F, E, R and L, respectively, and the second Fc polypeptide comprises substitutions of amino acids corresponding to amino acids at positions L234, L235E, G236 and K409 with F, E, R and R, respectively.

[0148] In another embodiment, one of the first Fc polypeptide and the second Fc polypeptide comprises substitutions of amino acids corresponding to amino acids at positions L234, L235, D265 and F405 with F, E, A and L, respectively, and the other Fc polypeptide comprises substitutions of amino acids corresponding to amino acids at positions L234, L235E, D265 and K409 with F, E, A and R, respectively.

[0149] In a further embodiment, the first Fc polypeptide comprises substitutions of amino acids corresponding to amino acids at positions L234, L235, D265 and F405 with F, E, A and L, respectively, and the second Fc polypeptide comprises substitutions of amino acids corresponding to amino acids at positions L234, L235E, D265 and K409 with F, E, A and R, respectively.

[0150] In a further embodiment, a multispecific antibody is disclosed, wherein the antibody is a bispecific antibody comprising a first heavy chain and a second heavy chain, and wherein the positions in both the first and second heavy chains corresponding to positions L234, L235, and G236 in a human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively, and wherein the position in the first heavy chain corresponding to F405 in a human IgG1 heavy chain according to Eu numbering is L, and the position in the second heavy chain corresponding to K409 in a human IgG1 heavy chain according to Eu numbering is R.

[0151] In a further embodiment, one of the first and second heavy chains comprises substitutions of amino acids corresponding to amino acids at positions L234, L235 and G236 with F, E and R, respectively, and the other heavy chain comprises substitutions of amino acids corresponding to amino acids at positions L234, L235 and D265 with F, E and A, respectively, where the amino acid positions are as defined by Eu numbering.

[0152] In still further embodiments, the antibody is a bispecific antibody comprising a first heavy chain and a second heavy chain, and wherein one heavy chain comprises substitutions of amino acids corresponding to amino acids at positions L234, L235, and G236 with F, E, and R, respectively, and the other heavy chain comprises substitutions of amino acids corresponding to amino acids at positions L234, L235, and D265 with F, E, and A, respectively, and wherein (i) the position in the first heavy chain corresponding to F405 of the human IgG1 heavy chain according to Eu numbering is L and the position in the second heavy chain corresponding to K409 of the human IgG1 heavy chain according to Eu numbering is R, or (ii) the position in the first heavy chain corresponding to K409 of the human IgG1 heavy chain according to Eu numbering is R and the position in the second heavy chain corresponding to F405 of the human IgG1 heavy chain according to Eu numbering is L.

[0153] In one embodiment, an antibody of the invention comprises or consists of the heavy chain sequences shown in SEQ ID NOs: 17 and 19 and the light chain sequences shown in SEQ ID NOs: 18 and 20.

[0154] The present invention further discloses a multispecific antibody as described herein, comprising (i) a first heavy chain and a first light chain linked via a disulfide bridge forming a first binding region that binds to FAPα, and (ii) a second heavy chain and a second light chain linked via a disulfide bridge forming a second binding region that binds to DR4, wherein i) the first heavy chain comprises the sequence set forth in SEQ ID NO: 17 and the first light chain comprises the sequence set forth in SEQ ID NO: 18, and ii) the second heavy chain comprises the sequence set forth in SEQ ID NO: 19 and the second light chain comprises the sequence set forth in SEQ ID NO: 20.

[0155] The constant region sequences listed in SEQ ID NOS: 21-26 and 70-72 do not contain a C-terminal lysine (K). However, in the naturally occurring sequences found in humans from which these Fc regions are derived, such a C-terminal lysine may be present as part of the open reading frame. During cell culture production of recombinant antibodies, this terminal lysine may be proteolytically cleaved by endogenous carboxypeptidases, resulting in a constant region with the same sequence but lacking the C-terminal lysine. For antibody manufacturing purposes, the DNA encoding this terminal lysine may be deleted from the sequence so that the antibody is produced without the lysine. Deleting the C-terminal lysine from the antibody-encoding sequence can improve antibody uniformity with respect to the presence of the C-terminal lysine. Antibodies produced from nucleic acid sequences that do or do not encode the terminal lysine are substantially identical in sequence and function. This is because, for example, when using antibodies produced in CHO-based production systems, the degree of C-terminal lysine processing is typically high (Dick, L. Wet et al. Biotechnol. Bioeng. 2008;100:1132-1143). It is therefore understood that antibodies according to the invention can be produced with or without encoding a C-terminal lysine as recited herein, and for manufacturing purposes, antibodies can be produced without such a C-terminal lysine.

[0156] In one embodiment, the multispecific antibody is a bispecific antibody comprising a first heavy chain and a second heavy chain, wherein the positions in both the first and second heavy chains corresponding to positions L234 and L235 in the human IgG1 heavy chain according to Eu numbering are F and E, respectively, and wherein (i) the position in the first heavy chain corresponding to F405 in the human IgG1 heavy chain according to Eu numbering is L and the position in the second heavy chain corresponding to K409 in the human IgG1 heavy chain according to Eu numbering is R, or (ii) the position in the first heavy chain corresponding to K409 in the human IgG1 heavy chain according to Eu numbering is R and the position in the second heavy chain corresponding to F405 in the human IgG1 heavy chain according to Eu numbering is L.

[0157] In a further embodiment, the multispecific antibody is a bispecific antibody comprising a first heavy chain and a second heavy chain, wherein the positions in both the first and second heavy chains corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to Eu numbering are F, E, and A, respectively, and wherein (i) the position in the first heavy chain corresponding to F405 in the human IgG1 heavy chain according to Eu numbering is L and the position in the second heavy chain corresponding to K409 in the human IgG1 heavy chain according to Eu numbering is R, or (ii) the position in the first heavy chain corresponding to K409 in the human IgG1 heavy chain according to Eu numbering is R and the position in the second heavy chain corresponding to F405 in the human IgG1 heavy chain according to Eu numbering is L.

[0158] In yet a further embodiment, the multispecific antibody is a bispecific antibody comprising a first heavy chain and a second heavy chain, wherein the positions in both the first and second heavy chains corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively, and wherein (i) the position in the first heavy chain corresponding to F405 in the human IgG1 heavy chain according to Eu numbering is L and the position in the second heavy chain corresponding to K409 in the human IgG1 heavy chain according to Eu numbering is R, or (ii) the position in the first heavy chain corresponding to K409 in the human IgG1 heavy chain according to Eu numbering is R and the position in the second heavy chain corresponding to F405 in the human IgG1 heavy chain according to Eu numbering is L.

[0159] In still further embodiments, the multispecific antibody is a bispecific antibody comprising a first heavy chain and a second heavy chain, and in which the positions corresponding to amino acids at positions L234, L235 and G236 are F, E and R, respectively, and the other Fc polypeptide comprises substitutions of amino acids corresponding to amino acids at positions L234, L235 and D265 with F, E and A, respectively, and in which (i) the position in the first heavy chain corresponding to F405 of the human IgG1 heavy chain according to Eu numbering is L and the position in the second heavy chain corresponding to K409 of the human IgG1 heavy chain according to Eu numbering is R, or (ii) the position in the first heavy chain corresponding to K409 of the human IgG1 heavy chain according to Eu numbering is R and the position in the second heavy chain corresponding to F405 of the human IgG1 heavy chain according to Eu numbering is L.

[0160] A multispecific antibody in the context of the present invention is a bispecific antibody comprising: (i) a first heavy chain and a first light chain comprising a FAPα-binding region, wherein the FAPα-binding region comprises a first heavy chain variable region and a first light chain variable region, the first heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 1, 2 and 3, respectively, and the first light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 4, 5 and 6, respectively; and (ii) a second heavy chain and a second light chain comprising a DR4-binding region, wherein the DR4-binding region comprises a second heavy chain variable region and a second light chain variable region, the second heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 8 and 9. and a second heavy chain and a second light chain, wherein the second heavy chain variable region (VL) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 10, 11, and 12, respectively, and the second light chain variable region (VL) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 10, 11, and 12, respectively; (iii) in which positions in both the first heavy chain and the second heavy chain corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively; and (iv) in which the first heavy chain has an L at a position corresponding to position F405 in the human IgG1 heavy chain according to Eu numbering, and the second heavy chain has an R at a position corresponding to K409 in the human IgG1 heavy chain according to Eu numbering.

[0161] A multispecific antibody in the context of the present invention may comprise or consist of a bispecific antibody comprising (i) the FAPα heavy chain sequence shown in SEQ ID NO: 17 and the FAPα light chain sequence shown in SEQ ID NO: 18, and (ii) the DR4 heavy chain sequence shown in SEQ ID NO: 19 and the DR4 light chain sequence shown in SEQ ID NO: 20.

[0162] In one embodiment, the multispecific antibody described herein is a bispecific, bivalent antibody. In a further embodiment, said multispecific antibody is a bispecific, bivalent antibody having monovalent binding to FAPα and monovalent binding to DR4.

[0163] In alternative embodiments, the multispecific antibody according to the present invention is not a classical full-length antibody comprising an Fc region. For example, in one embodiment, the multispecific antibody is an antibody fragment. In further embodiments, (i) the FAPα-binding region and / or the DR4-binding region is a Fab, (ii) the FAPα-binding region and / or the DR4-binding region is an scFv, (iii) the FAPα-binding region is a Fab and the DR4-binding region is an scFv, or (iv) the FAPα-binding region is an scFv and the DR4-binding region is a Fab.

[0164] Binding and transactivation The multispecific antibody, e.g., bispecific antibody, described herein, capable of binding to FAPα and DR4, e.g., human FAPα and human DR4, can advantageously target DR4 and FAPα-positive cells, e.g., CAFs, on tumor cells, thereby specifically inducing apoptosis of tumor cells. In one embodiment, the multispecific antibody is a transactivating antibody. In a further embodiment, the multispecific antibody induces regulated DR4-mediated activation by binding to FAPα, such as transbinding to FAPα, leading to apoptosis, for example. Thus, the multispecific antibody is capable of FAPα-dependent DR4 transactivation. This means that the apoptotic effect induced by DR4 activation upon binding of the multispecific antibody to DR4 is only observed when the antibody binds to both DR4 and FAPα.

[0165] As mentioned above, preferably the multispecific antibodies according to the invention lack or have reduced Fc-mediated effector function and further the antibodies i) capable of binding to FAPα-expressing fibroblasts, such as cancer-associated fibroblasts (CAFs) as described in Example 2 herein; ii) capable of binding to DR4-expressing human tumor cell lines as described in Example 3 herein; iii) capable of binding in vitro in the presence of one or more DR4-expressing human tumor cell lines selected from the group consisting of DLD-1, A549, HCT-116, HCT-15, MDA-MB-231 and PANC-1 when assayed, for example, as described in Example 3 herein; iv) is capable of mediating cell death in a DR4-expressing human tumor cell line in the presence of FAPα-expressing cells, such as FAPα-expressing fibroblasts, when assayed as described in Example 10 herein; v) is capable of mediating concentration-dependent cell death in a DR4-expressing human tumor cell line in the presence of FAPα-expressing cells, such as FAPα-expressing fibroblasts, when assayed as described in Example 10 herein; vi) is capable of mediating concentration-dependent cell death in one or more human DR4-expressing tumor cell lines selected from the group consisting of DLD-1 and MDA-MB-231 in the presence of FAPα-expressing fibroblasts, when assayed as described in Example 10 herein; vii) is capable of inducing caspase-8 activation in one or more human DR4-expressing tumor cell lines selected from the group consisting of DLD-1 and MDA-MB-231 when assayed in the presence of FAPα-expressing fibroblasts as described in Example 10 herein; viii) can kill CRC-derived organoids in the presence of FAPα-expressing CAFs, for example, when assayed as described in Example 11 herein; ix) capable of antitumor activity against pancreatic and gastric tumors, e.g., when assayed as described in Example 12 herein; x) capable of antitumor and anti-metastatic activity, e.g., when assayed as described in Example 13 herein; xi) capable of antitumor and anti-metastatic activity against CRC, e.g., when assayed as described in Example 13 herein; xii) does not exhibit hepatotoxicity when assayed, for example, as described in Example 14 herein; xiii) is unable to bind C1q, e.g., when assayed as described in Example 18 herein; xiv) is unable to bind to an FcγR, e.g., FcγRIa, FcγRIIa, FcγRIIb, and / or FcγRIIIa, e.g., when assayed as described herein in Example 18; xv) is capable of binding to FcRn, e.g., when assayed as described in Example 18 herein; and / or xvi) exhibits pharmacokinetic properties similar to wild-type IgG1, for example, when assayed as described in Example 19 herein.

[0166] Furthermore, the antibodies according to the invention are capable of inducing transactivation-mediated cell death, and the cytotoxicity is i) providing transfected NIH / 3T3 cells to express the FAPα mature polypeptide; ii) providing DR4-expressing tumor cells, such as MDA-MB-231 or DLD-1; iii) combining the NIH / 3T3 with the DR4-expressing tumor cells at a ratio of 1:2 in the number of NIH / 3T3 cells to selected tumor cells; iv) providing the antibody to the sample in a dilution series ranging from, for example, 0.28 ng / mL to 14,400 ng / mL; v) Incubating the sample obtained in step iv) for example at 37°C for 72 hours, followed by vi) assessing survival of DR4-expressing tumor cells; vii) determining the percentage of viable cells for each diluted sample, for example using a luminescence readout; and viii) determining the percentage of viable cells; The cells are evaluated in an in vitro survival assay including:

[0167] In one embodiment, the antibody is capable of inducing cell death, e.g., transactivation-mediated cell death, by at least 20% at an antibody concentration of 28 ng / ml and / or by at least 45%, e.g., at least 50%, at an antibody concentration of 1800 ng / ml.

[0168] In a further embodiment, the multispecific antibodies according to the invention do not induce detectable hepatotoxicity, which can be monitored, for example, by measuring lactate dehydrogenase, e.g., on day 4, and / or intracellular adenosine triphosphate, e.g., on day 6 and / or 7, after exposing liver spheroids consisting of primary human hepatocytes and non-parenchymal liver cell types to the multispecific antibody.

[0169] Nucleic Acid Constructs and Expression Vectors A further aspect of the present invention provides a nucleic acid construct encoding an antibody defined herein, or a combination of nucleic acid constructs. For example, in one embodiment, the combination of nucleic acid constructs comprises a first construct encoding a first heavy chain, a second construct encoding a second heavy chain, a third construct encoding a first light chain, and a fourth construct encoding a second light chain. Alternatively, the combination of nucleic acid constructs comprises a first construct encoding a first heavy chain and a first light chain, and a second construct encoding a second heavy chain and a second light chain.

[0170] Another aspect of the present invention provides an expression vector or a combination of expression vectors comprising one or more nucleic acid constructs described herein.The expression vector in the context of the present invention can be any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (nucleic acid sequences comprising a set of appropriate expression control elements).Examples of such vectors include SV40 derivatives, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from a combination of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid encoding the anti-FAPα antibody and / or the nucleic acid encoding the anti-DR4 antibody is contained in a naked DNA or RNA vector, e.g., a linear expression element (e.g., as described in Sykes and Johnston, Nat Biotech 17, 355-59 (1997)), a compacted nucleic acid vector (e.g., as described in U.S. Pat. No. 6,077,835 and / or WO 00 / 70087), a plasmid vector, e.g., pBR322, pUC 19 / 18, or pUC 118 / 119, the minimal size nucleic acid vector "midge" (e.g., as described in Schakowski et al., Mol Ther 3, 793-800 (2001)), or a precipitated nucleic acid vector construct, e.g., a CaPO4 precipitated construct (e.g., as described in WO 00 / 46147; Benvenisty and Reshef, PNAS USA 83,9551-55 (1986), Wigler et al., Cell 14,725 (1978), and Coraro and Pearson, Somatic Cell Genetics 7,603 (1981). Such nucleic acid vectors and methods for their use are well known in the art (see, e.g., U.S. Pat. Nos. 5,589,466 and 5,973,972).

[0171] In one embodiment, the vector is suitable for expressing an anti-FAPα antibody and / or an anti-DR4 antibody in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503-5509 (1989)), and pET vectors (Novagen, Madison WI).

[0172] Additionally or alternatively, the expression vector may be a vector suitable for expression in yeast systems. Any vector suitable for expression in yeast systems may be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH (reviewed in F. Ausubel et al., eds., Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987) and Grant et al., Methods in Enzymol 153, 516-544 (1987)).

[0173] The nucleic acid construct and / or vector can also include a nucleic acid sequence encoding a secretion / localization sequence, thereby targeting a polypeptide, e.g., a nascent polypeptide chain, to the periplasmic space or into the cell culture medium. Such sequences are known in the art and include secretory leader or signal peptides, organelle targeting sequences (e.g., nuclear localization sequences, ER retention signals, mitochondrial import sequences, chloroplast import sequences), membrane localization / anchor sequences (e.g., stop transport sequences, GPI anchor sequences), etc.

[0174] The nucleic acid and / or expression vector can also include a nucleic acid sequence encoding a secretion / localization sequence, thereby targeting a polypeptide, e.g., a nascent polypeptide chain, to the periplasmic space or cell culture medium. Such sequences are known in the art and include secretory leaders or signal peptides. The nucleic acid and / or expression vector can include any suitable elements that facilitate expression, i.e., transcription and / or translation of the nucleic acid, so that the components of the (bispecific) antibody are expressed. The nucleic acid and / or vector is associated with any suitable promoter, enhancer, and other expression-enhancing elements. Examples of such elements include a strong expression promoter (e.g., the human CMV IE promoter / enhancer and RSV, SV40, SL33, MMTV, and HIV LTR promoters), an effective poly(A) termination sequence, an origin of replication for the plasmid product in E. coli, an antibiotic resistance gene as a selectable marker, and / or a convenient cloning site (e.g., a polylinker). The nucleic acid can also include an inducible promoter, as opposed to a constitutive promoter such as CMV IE.

[0175] In a further aspect, the present invention provides a composition comprising a nucleic acid construct or a combination of nucleic acid constructs as defined herein.

[0176] Delivery Vehicle In a further aspect, the present invention relates to the administration of a nucleic acid construct encoding an antibody of the present invention for in vivo expression. For in vivo expression of a nucleic acid encoding an antibody, the nucleic acid is typically administered in a form suitable for the nucleic acid to enter the cells of a subject. There are various methods of nucleic acid delivery for in vivo expression, including both mechanical and chemical methods. For example, such methods may include electroporation or tattooing the nucleic acid onto the skin (Patel et al., 2018, Cell Reports 25, 1982-1993). Other suitable methods for administering nucleic acids to a subject include administering the nucleic acid in a suitable formulation.

[0177] Therefore, the present invention also relates to a delivery vehicle comprising the nucleic acid construct described herein. In one embodiment, the delivery vehicle can be a particle. In some embodiments, the delivery vehicle can be a lipid formulation. The lipid of the formulation can be a particle, such as a lipid nanoparticle (LNP). The nucleic acid or nucleic acid combination of the present invention can be encapsulated within the particle, for example, within the LNP. Various lipid formulations suitable for administering nucleic acids to a subject for in vivo expression are well known to those skilled in the art. For example, the lipid formulation can typically include a lipid, an ionizable amino lipid, a PEG-lipid, cholesterol, or any combination thereof.

[0178] The various forms and methods for preparing lipid formulations suitable for administering nucleic acid to subject for expressing therapeutic antibody are well known in the art.The example of such lipid formulation includes but is not limited to those described in US Patent No. 20180170866 (Arcturus), European Patent No. 2391343 (Arbutus), International Publication No. 2018 / 006052 (Protiva), International Publication No. 2014152774 (Shire Human Genetics), European Patent No. 2972360 (Translate Bio), US Patent No. 10195156 (Moderna) and US Patent No. 20190022247 (Acuitas).

[0179] Thus, in a further aspect, the present invention relates to (a) one or more nucleic acid constructs according to the invention or a delivery vehicle according to the invention for use as a medicament, preferably for use in the treatment of cancer, such as the treatment of solid cancers.

[0180] Cells and host cells In a further aspect, the present invention provides a recombinant host cell capable of producing a multispecific antibody as described herein, comprising one or more nucleic acid constructs encoding the antibody as defined herein above, or an expression vector as defined herein above. It should be understood that the cell may be obtained by transfecting a host cell, e.g., a recombinant host cell, with said nucleic acid construct or expression vector. In one embodiment, the host cell is an isolated host cell.

[0181] The host cells may be of human origin, e.g., human embryonic kidney (HEK) cells, such as HEK / Expi cells, or may be of rodent origin, e.g., Chinese hamster ovary cells, such as CHO / N50 cells or CHO cells. Additionally, the host cells may be of bacterial origin.

[0182] The host cell may contain a nucleic acid sequence encoding the antibody of the present invention or a portion thereof stably integrated into the cellular genome. Alternatively, the cell may contain a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element comprising a sequence encoding the expression of the anti-FAPα antibody and / or anti-DR4 antibody of the present invention or a portion thereof. In particular, the host cell may contain a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element comprising a sequence encoding the expression of the anti-FAPα antibody and / or anti-DR4 antibody or a portion thereof.

[0183] Compositions, (medical) uses and therapeutic applications Furthermore, the present invention provides a composition comprising an antibody as defined herein. Preferably, such a composition is a pharmaceutical composition, i.e. the antibody is in a pharmaceutically acceptable carrier.

[0184] Pharmaceutical compositions can be formulated according to conventional techniques, such as those described in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed. Mack Publishing Co., Easton, PA, 1995. Pharmaceutical compositions of the present invention can include, for example, diluents, fillers, salts, buffers, surfactants (e.g., non-ionic surfactants such as Tween-20 or Tween-80), stabilizers (e.g., sugars, or protein-free amino acids), preservatives, tissue fixatives, solubilizing agents, and / or other materials suitable for inclusion in pharmaceutical compositions.

[0185] The pharmaceutical composition or multispecific antibody may be administered in a therapeutically effective amount by any suitable route and mode, hi one embodiment, the pharmaceutical composition and / or multispecific antibody is administered by intravenous injection or infusion.

[0186] In a further aspect, the multispecific antibody, nucleic acid construct(s), delivery vehicle, composition or pharmaceutical composition described herein is for use as a medicament.

[0187] In still a further aspect, the multispecific antibody, nucleic acid construct(s), delivery vehicle, composition or pharmaceutical composition described herein is for use in the treatment of a disease such as cancer.

[0188] In particular, the bispecific antibodies of the present invention may be used in the treatment of various forms of cancer.

[0189] Further disclosed herein are multispecific antibodies, nucleic acid constructs, delivery vehicles, compositions or pharmaceutical compositions for use in the treatment of primary tumors and / or for use in the prevention and / or treatment of metastases.

[0190] In one aspect, the present invention relates to a multispecific antibody, one or more nucleic acid constructs, a delivery vehicle, or a pharmaceutical composition for use in treating a cancer that is a solid cancer. In one embodiment, the solid cancer is a malignant solid cancer, such as a malignant solid tumor. In a further embodiment, the malignant solid tumor is an advanced solid tumor and / or a metastatic solid tumor. In a further embodiment, the malignant solid tumor is a metastatic solid tumor. In one embodiment, the solid cancer is a metastatic cancer. In a further aspect, the present invention relates to the use of a multispecific antibody, one or more nucleic acid constructs, a delivery vehicle, or a pharmaceutical composition. In one embodiment, the cancer is a carcinoma. Examples of cancers that can be treated are cancers selected from the group of colorectal cancer (CRC), such as colorectal adenocarcinoma, breast cancer, such as triple-negative breast cancer, pancreatic cancer, such as pancreatic ductal adenocarcinoma, gastric cancer, and lung cancer, such as non-small cell lung cancer. In particular, the cancer is selected from the group of pancreatic cancer, gastric cancer, and CRC. In further embodiments, the cancer is selected from the group consisting of colorectal cancer (CRC), breast cancer, e.g., triple-negative breast cancer (TNBC), pancreatic cancer, e.g., pancreatic ductal adenocarcinoma (PDAC), esophagogastric cancer, e.g., gastric cancer and esophageal cancer, head and neck squamous cell carcinoma (HNSCC), cervical cancer, and lung cancer, e.g., non-small cell lung cancer (NSCLC).

[0191] In one aspect, the invention provides a method for treating cancer in a subject, the method comprising administering a therapeutically effective amount of a multispecific antibody of the invention. In a further embodiment, the invention provides a method for treating a disorder in a subject involving cells expressing DR4 that are in proximity to cells expressing FAPα, the method comprising administering a therapeutically effective amount of a multispecific antibody of the invention. DR4-expressing cells in proximity to FAPα-expressing cells allow trans-binding of the multispecific antibody according to the invention.

[0192] As described above, a preferred disease contemplated in the methods and uses according to the present invention is cancer. The cancer is most preferably characterized by the expression of DR4. DR4 expression in cancer can be easily determined using methods known in the art, such as PCR, immunostaining, or FACS analysis, i.e., detecting the expression of DR4 transcripts and / or proteins. The antibodies described herein capable of binding to human DR4 can be used, for example, for immunostaining and / or FACS analysis. Furthermore, FAPα-expressing cells, such as CAFs, should preferably be detected in the TME. Thus, in one embodiment, the TME contains CAFs, and preferably, the CAFs express FAPα. FAPα expression in the TME can be easily determined using methods known in the art, such as PCR, immunostaining, or FACS analysis, i.e., detecting the expression of DR4 transcripts and / or proteins. Preferably, the multispecific antibody, one or more nucleic acid constructs, delivery vehicle, or pharmaceutical composition is used for treatment when the cancer expresses DR4, the tumor microenvironment contains CAFs, and the CAFs express FAPα.

[0193] In a further embodiment, patients diagnosed with cancer can be subjected to an assessment of DR4 expression in cancer cells and FAPα-expressing cells in the TME, and if DR4 and FAPα, which may range from low to high, are detected, such patients can be selected for treatment with an antibody according to the present invention. However, it may not be necessary to include such assessments when selecting patients for treatment.

[0194] In a further aspect, the present invention relates to a method for treating cancer, comprising administering a therapeutically effective amount of a multispecific antibody defined herein, one or more nucleic acid constructs described herein, a delivery vehicle described herein, a composition described herein, or a pharmaceutical composition described herein to a subject in need thereof. In particular, the method may be for treating solid cancers, primary tumors, and / or metastases. Examples of cancers to be treated may be selected from the group consisting of CRC, breast cancer, pancreatic cancer, gastric cancer, and lung cancer. In particular, the cancer is selected from the group consisting of pancreatic cancer, gastric cancer, and colorectal cancer.

[0195] In a still further aspect, the invention relates to the use of a multispecific antibody as described herein in the manufacture of a medicament for treating cancer.

[0196] kit The present invention further provides a kit of parts comprising the antibody disclosed above, e.g. for use as a companion diagnostic / for identifying patients within a patient population who have a propensity to respond to treatment with an antibody as defined herein above, or for predicting the efficacy or anti-tumor activity of said antibody when used in treating patients, said kit comprising the antibody as defined herein above, and instructions for use of said kit.

[0197] In one embodiment, the present invention provides a kit for diagnosing cancer, comprising a container containing a multispecific FAPαxDR4 antibody and one or more reagents for detecting crosslinking of FAPα-expressing cells and DR4-expressing cells. The reagents may include, for example, fluorescent tags, enzyme tags, or other detectable tags. The reagents may also include secondary or tertiary antibodies, or reagents for enzymatic reactions that produce visualized products.

[0198] In a further aspect, the present invention provides a diagnostic composition comprising an antibody as defined herein. The diagnostic composition may further comprise a dilution buffer.

[0199] In a further aspect, the present invention relates to a method for detecting whether administration of a multispecific antibody according to any one of the embodiments disclosed herein results in cross-linking between FAPα- and DR4-expressing cells in a sample derived from a patient, the method comprising the steps of (i) contacting the sample with a multispecific antibody according to any one of the embodiments disclosed herein under conditions that allow the formation of a complex between said bispecific antibody and the FAPα- and DR4-expressing cells, and (ii) analyzing whether a complex has been formed.

[0200] FAPα antibody In a further aspect, the present invention relates to a monospecific antibody and provides an anti-FAPα antibody comprising at least one FAPα-binding region, wherein the FAPα-binding region comprises a heavy chain variable region (VH) comprising three complementarity-determining regions, CDR1, CDR2, and CDR3, present within the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable region (VL) comprising three complementarity-determining regions, CDR1, CDR2, and CDR3, present within the amino acid sequence set forth in SEQ ID NO: 14. In one embodiment, the FAPα-binding region is capable of binding to FAPα.

[0201] In a further aspect, the present invention provides an anti-FAPα antibody, which relates to a monospecific antibody and comprises at least one FAPα binding region capable of binding to FAPα, wherein the FAPα binding region comprises a heavy chain variable region (VH) comprising three complementarity determining regions CDR1, CDR2 and CDR3 present within the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable region (VL) comprising three complementarity determining regions CDR1, CDR2 and CDR3 present within the amino acid sequence set forth in SEQ ID NO: 14.

[0202] In a further embodiment, an anti-FAPα antibody in the context of the present invention relates to an anti-FAPα antibody in which the apparent affinity of the anti-FAPα antibody for monovalent binding, e.g., to human FAPα expressed by human lung fibroblasts, is higher, e.g., at least 5-fold higher, e.g., at least 10-fold higher, as measured, e.g., as exemplified in Example 2, compared to the monovalent binding reference antibody anti-FAP5. As known to those skilled in the art, EC 50 A lower value indicates a higher apparent affinity.

[0203] In a further embodiment, the maximum binding (max gMFI) of the anti-FAPα antibody at a concentration of 10 μg / mL is higher, for example at least 25% higher, for example at least 40% higher, compared to the reference antibody anti-FAP5, when the anti-FAPα antibody and the anti-FAP5 antibody bind monovalently to human FAPα expressed by, for example, human lung fibroblasts, for example, as measured as illustrated in Example 2.

[0204] In a further embodiment, the apparent affinity of the anti-FAPα antibody for bivalent binding, e.g., to human FAPα expressed by human lung fibroblasts, is higher, e.g., at least 5-fold higher, e.g., at least 10-fold higher, as measured, e.g., as exemplified in Example 2, relative to the bivalent binding reference antibody anti-FAP5. As known to those skilled in the art, EC 50 A lower value indicates a higher apparent affinity.

[0205] Further disclosed herein is an anti-FAPα antibody, wherein the anti-FAPα antibody comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 1, 2, and 3, respectively. Also disclosed herein is an anti-FAPα antibody, wherein the anti-FAPα antibody comprises a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 4, 5, and 6, respectively. In one embodiment, the anti-FAPα antibody comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 1, 2, and 3, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 4, 5, and 6, respectively. The CDR regions from the variable heavy and light chain regions are annotated according to IMGT (see Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, Developmental and Comparative Immunology, 27(1), 55-77 (2003)).

[0206] The present disclosure further provides an anti-FAPα antibody, wherein the VH sequence of the FAPα-binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. The present disclosure also provides an anti-FAPα antibody, wherein the VL sequence of the FAPα-binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In one embodiment, the VH sequence of the FAPα-binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 13, and the VL sequence of the FAPα-binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 14.

[0207] In a still further embodiment, said VH sequence of the FAPα-binding region set forth in SEQ ID NO: 13 comprises up to 10 substitutions, such as up to 9 substitutions, for example up to 8 substitutions, such as up to 7 substitutions, for example up to 6 substitutions, such as up to 5 substitutions, for example up to 4 substitutions, for example up to 3 substitutions, such as up to 2 substitutions, for example up to 1 substitution. In a still further embodiment, said VL sequence of the FAPα-binding region set forth in SEQ ID NO: 14 comprises up to 10 substitutions, such as up to 9 substitutions, for example up to 8 substitutions, such as up to 7 substitutions, for example up to 6 substitutions, such as up to 5 substitutions, for example up to 4 substitutions, for example up to 3 substitutions, such as up to 2 substitutions, for example up to 1 substitution. In a still further embodiment, the VH sequence and VL sequence of an anti-FAPα antibody differ only in the framework regions. In a still further embodiment, said VH sequence of the FAP alpha binding region differs from SEQ ID NO: 13 by up to 10 substitutions, such as up to 9 substitutions, for example up to 8 substitutions, such as up to 7 substitutions, for example up to 6 substitutions, such as up to 5 substitutions, for example up to 4 substitutions, such as up to 3 substitutions, for example up to 2 substitutions, such as up to 1 substitution. In a still further embodiment, said VL sequence of the FAP alpha binding region differs from SEQ ID NO: 14 by up to 10 substitutions, such as up to 9 substitutions, for example up to 8 substitutions, such as up to 7 substitutions, for example up to 6 substitutions, such as up to 5 substitutions, for example up to 4 substitutions, for example up to 3 substitutions, such as up to 2 substitutions, for example up to 1 substitution.

[0208] The anti-FAPα antibody according to the present invention may be either monovalent or bivalent. In one embodiment, the anti-FAPα antibody is monovalent. In another embodiment, the anti-FAPα antibody is a bivalent antibody having two antigen-binding regions capable of binding to human FAPα, and preferably, the two antigen-binding regions have the same variable region sequence.

[0209] In the context of the present invention, an anti-FAPα antibody may contain one or more substitutions in the first and / or second heavy chain, thereby comprising an Fc region comprising different first and second CH3 regions, resulting in heterodimeric interactions between said first and second CH3 regions, or an Fc region comprising similar first and second CH3 regions, resulting in homodimeric interactions between said first and second CH3 regions. The first and / or second heavy chain of an anti-FAPα antibody may also contain modifications in the Fc region to render the antibody inactive or non-activating, similar to the substitutions and modifications described for multispecific antibodies above. Accordingly, the present disclosure further provides an anti-FAPα antibody, (i) wherein the anti-FAPα antibody comprises a first heavy chain and a second heavy chain; (ii) wherein each of the first heavy chain and the second heavy chain comprises at least a hinge region, a CH2 region, and a CH3 region; and (iii) wherein in the first heavy chain, at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (according to EU numbering) is substituted, and in the second heavy chain, at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (according to EU numbering) is substituted. The present disclosure also further provides an anti-FAPα antibody described herein, (i) wherein the anti-FAPα antibody comprises a first heavy chain and a second heavy chain; (ii) wherein each of the first heavy chain and the second heavy chain comprises at least a hinge region, a CH2 and a CH3 region; and (iii) wherein in the first heavy chain, at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 (in a human IgG1 heavy chain according to Eu numbering) has been substituted, and in the second heavy chain, at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 (in a human IgG1 heavy chain according to Eu numbering) has been substituted.In a further embodiment, the first heavy chain and the second heavy chain are substituted at the same positions. In a still further embodiment, an anti-FAPα antibody is provided, wherein (i) the amino acid at the position corresponding to F405 (according to Eu numbering) is L, or (ii) the amino acid at the position corresponding to K409 (according to Eu numbering) is R. In a still further embodiment, an anti-FAPα antibody is provided, wherein (i) the amino acid at the position corresponding to F405 (according to Eu numbering) is L. In a still further embodiment, (i) the amino acid at the position corresponding to F405 (in the human IgG1 heavy chain according to Eu numbering) is L, or (ii) the amino acid at the position corresponding to K409 (in the human IgG1 heavy chain according to Eu numbering) is R.

[0210] The present disclosure further provides an anti-FAPα antibody, which comprises a first heavy chain and a second heavy chain, and wherein one or both heavy chains are modified to reduce the degree of Fc-mediated effector function induced by the antibody compared to an identical antibody except for comprising unmodified first and second heavy chains.

[0211] The anti-FAPα antibody may comprise a first heavy chain and a second heavy chain, wherein in at least one of said first heavy chain and said second heavy chain, one or more amino acids at positions corresponding to positions L234, L235, G236, D265, N297, and P331 in a human IgG1 heavy chain according to Eu numbering are not L, L, G, D, N, and P, respectively. In one embodiment, the anti-FAPα antibody is one in which positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to Eu numbering are F and E in said first heavy chain and said second heavy chain, respectively. In yet a further embodiment, positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to Eu numbering are F, E, and A, respectively, in said first heavy chain and / or said second heavy chain. In still further embodiments, the positions corresponding to positions L234, L235 and G236 in a human IgG1 heavy chain according to Eu numbering are F, E and R in said first heavy chain and said second heavy chain, respectively.

[0212] The present disclosure further provides an anti-FAPα antibody, wherein the anti-FAPα antibody comprises a first heavy chain and a second heavy chain, and wherein in both the first heavy chain and the second heavy chain, positions corresponding to positions L234, L235, and G236 in a human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively, and wherein (i) the position corresponding to F405 in a human IgG1 heavy chain according to Eu numbering is L, or (ii) the position in the first heavy chain corresponding to K409 in a human IgG1 heavy chain according to Eu numbering is R.

[0213] An anti-FAPα antibody in the context of the present invention may comprise a FAPα binding region in which (i) the heavy chain variable region (VH) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 1, 2, and 3, respectively, and the light chain variable region (VL) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 4, 5, and 6, respectively; (ii) wherein the anti-FAPα antibody comprises a first heavy chain and a second heavy chain, and the positions in both the first heavy chain and the second heavy chain corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively; and (iii) the position in the first heavy chain corresponding to position F405 in the human IgG1 heavy chain according to Eu numbering is L, or the position in the first heavy chain corresponding to position K409 in the human IgG1 heavy chain according to Eu numbering is L. In one embodiment, the antibody comprises a FAPα binding region, (i) wherein the heavy chain variable region (VH) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 1, 2, and 3, respectively, and the light chain variable region (VL) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 4, 5, and 6, respectively; (ii) wherein the anti-FAPα antibody comprises a first heavy chain and a second heavy chain, and wherein the positions in both the first heavy chain and the second heavy chain corresponding to positions L234, L235, and G236 in a human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively; and (iii) wherein the position in the first heavy chain corresponding to position F405 in a human IgG1 heavy chain according to Eu numbering is L.

[0214] An anti-FAPα antibody in the context of the present invention may comprise the heavy chain sequence shown in SEQ ID NO:17 and the light chain sequence shown in SEQ ID NO:18.

[0215] The present disclosure further provides an anti-FAPα antibody, wherein the EC 50 When binding to human lung fibroblasts or CAFs, for example, when assayed as described in Example 2 herein, the antibody has a cytotoxicity of 0.005 to 0.1 μg / mL, e.g., a cytotoxicity of 0.012 to 0.2 μg / mL.

[0216] The present disclosure further provides an anti-FAPα antibody, wherein the EC 50 When bivalently bound to human lung fibroblasts or CAFs, for example, when assayed as described in Example 2 herein, the antibody has a mAb concentration in the range of 0.005 to 0.1 μg / mL, e.g., in the range of 0.01 to 0.05 μg / mL, e.g., in the range of 0.012 to 0.2 μg / mL.

[0217] DR4 antibody In a further aspect, the present invention relates to a monospecific antibody and provides an anti-DR4 antibody comprising at least one DR4 binding region, wherein the DR4 binding region comprises a heavy chain variable region (VH) comprising three complementarity determining regions CDR1, CDR2 and CDR3 present within the amino acid sequence set forth in SEQ ID NO: 15, and a light chain variable region (VL) comprising three complementarity determining regions CDR1, CDR2 and CDR3 present within the amino acid sequence set forth in SEQ ID NO: 16. In one embodiment, the DR4 binding region is capable of binding to DR4.

[0218] In a further aspect, the present invention provides an anti-DR4 antibody, which relates to a monospecific antibody and comprises at least one DR4 binding region capable of binding to DR4, wherein the DR4 binding region comprises a heavy chain variable region (VH) comprising three complementarity determining regions CDR1, CDR2 and CDR3 present within the amino acid sequence set forth in SEQ ID NO: 15, and a light chain variable region (VL) comprising three complementarity determining regions CDR1, CDR2 and CDR3 present within the amino acid sequence set forth in SEQ ID NO: 16.

[0219] An anti-DR4 antibody in the context of the present invention relates in a further embodiment to an anti-DR4 antibody, for example an anti-DR4 antibody that monovalently binds to human DR4 expressed by the human tumor cell line MDA-MB-231, the apparent affinity of which is (i) e.g., at least two-fold higher, e.g., at least three-fold higher, compared to the monovalently binding reference antibody IgG1-DR4-chCTB007, e.g., when measured as exemplified in Example 3; and / or (ii) is at least 5-fold higher, such as at least 8-fold higher, compared to the monovalent binding reference antibody IgG1-DR4-T1014A04, e.g., when measured as exemplified in Example 3.

[0220] As known to those skilled in the art, EC 50 A lower value indicates a higher apparent affinity.

[0221] In a further embodiment, the maximum binding (max gMFI) of said anti-DR4 antibody at a concentration of 90 μg / mL is: (i) the monovalent binding of said anti-DR4 antibodies and said IgG1-DR4-chCTB007 antibodies to human DR4, e.g., expressed by the human tumor cell line MDA-MB-231, compared to the reference antibody IgG1-DR4-chCTB007, e.g., at least two-fold higher, e.g., at least three-fold higher, e.g., as measured as exemplified in Example 3; and / or (ii) monovalent binding of said anti-DR4 antibodies and said IgG1-DR4-T1014A04 antibodies to human DR4, e.g., expressed by the human tumor cell line MDA-MB-231, e.g., as measured as exemplified in Example 3, e.g., at least 5-fold higher, e.g., at least 9-fold higher, compared to the reference antibody IgG1-DR4-T1014A04.

[0222] In a further embodiment, the apparent affinity of the anti-DR4 antibody for bivalent binding to human DR4, e.g., expressed by the human tumor cell line MDA-MB-231, is higher, e.g., at least 2-fold higher, e.g., at least 5-fold higher, as measured, e.g., as illustrated in Example 3, compared to the bivalent binding reference antibody anti-IgG1-DR4-T1014A04. As known to those skilled in the art, EC 50 A lower value indicates a higher apparent affinity.

[0223] In a further embodiment, the maximum binding (max gMFI) of said anti-DR4 antibody at a concentration of 90 μg / mL is, e.g. at least 2-fold higher, e.g. at least 3-fold higher, compared to the reference antibody IgG1-DR4-T1014A04, when said anti-DR4 antibody and said IgG1-DR4-T1014A04 antibody monovalently binds to human DR4, e.g. expressed by the human tumor cell line MDA-MB-231, e.g. measured as exemplified in Example 3.

[0224] Further disclosed herein is an anti-DR4 antibody, wherein the DR4 binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 8 and 9, respectively. Also disclosed herein is an anti-DR4 antibody, wherein the DR4 binding region comprises a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, 11 and 12, respectively. In one embodiment, the DR4 binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 8 and 9, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, 11 and 12, respectively. The CDR regions from the variable heavy and light chain regions are annotated according to IMGT (see Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, Developmental and Comparative Immunology, 27(1), 55-77 (2003)).

[0225] The present disclosure further provides anti-DR4 antibodies, wherein the VH sequence of the DR4 binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. The present disclosure also provides anti-DR4 antibodies, wherein the VL sequence of the DR4 binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 16. In one embodiment, the VH sequence of the DR4 binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 15, wherein the VL sequence of the DR4 binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 16.

[0226] In a still further embodiment, said VH sequence of the DR4 binding region set forth in SEQ ID NO: 15 comprises up to 10 substitutions, such as up to 9 substitutions, for example up to 8 substitutions, such as up to 7 substitutions, for example up to 6 substitutions, such as up to 5 substitutions, for example up to 4 substitutions, such as up to 3 substitutions, for example up to 2 substitutions, such as up to 1 substitution. In a still further embodiment, said VL sequence of the DR4 binding region set forth in SEQ ID NO: 16 comprises up to 10 substitutions, such as up to 9 substitutions, for example up to 8 substitutions, such as up to 7 substitutions, for example up to 6 substitutions, such as up to 5 substitutions, for example up to 4 substitutions, such as up to 3 substitutions, for example up to 2 substitutions, such as up to 1 substitution. In a still further embodiment, the VH and VL sequences differ only in the framework regions. In a still further embodiment, said VH sequence of a DR4 binding region differs from SEQ ID NO: 15 by up to 10 substitutions, such as up to 9 substitutions, for example up to 8 substitutions, such as up to 7 substitutions, for example up to 6 substitutions, such as up to 5 substitutions, for example up to 4 substitutions, such as up to 3 substitutions, for example up to 2 substitutions, such as up to 1 substitution. In a still further embodiment, said VL sequence of a DR4 binding region differs from SEQ ID NO: 16 by up to 10 substitutions, such as up to 9 substitutions, for example up to 8 substitutions, such as up to 7 substitutions, for example up to 6 substitutions, such as up to 5 substitutions, for example up to 4 substitutions, for example up to 3 substitutions, such as up to 2 substitutions, for example up to 1 substitution.

[0227] The anti-DR4 antibody according to the present invention may be either monovalent or bivalent. In one embodiment, the anti-DR4 antibody is monovalent. In another embodiment, the anti-DR4 antibody is a bivalent antibody having two antigen-binding regions capable of binding to human DR4, and preferably, the two antigen-binding regions have the same variable region sequence.

[0228] In the context of the present invention, an anti-DR4 antibody may comprise one or more substitutions in the first and / or second heavy chain, thereby comprising an Fc region comprising different first and second CH3 regions, resulting in heterodimeric interactions between said first and second CH3 regions, or an Fc region comprising similar first and second CH3 regions, resulting in homodimeric interactions of said first and second CH3 regions. The first and / or second heavy chain of an anti-DR4 antibody may also comprise modifications in the Fc region to render the antibody inactive or non-activating, similar to the substitutions and modifications described for multispecific antibodies above. Accordingly, the present disclosure further provides an anti-DR4 antibody, (i) wherein the anti-DR4 antibody comprises a first heavy chain and a second heavy chain; (ii) wherein each of the first heavy chain and the second heavy chain comprises at least a hinge region, a CH2 region, and a CH3 region; and (iii) wherein in the first heavy chain, at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (according to EU numbering) has been substituted; and in the second heavy chain, at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (according to EU numbering) has been substituted. The present disclosure also further provides an anti-DR4 antibody, (i) wherein the anti-DR4 antibody comprises a first heavy chain and a second heavy chain, (ii) wherein each of the first heavy chain and the second heavy chain comprises at least a hinge region, a CH2 region, and a CH3 region, and (iii) wherein in the first heavy chain, at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (in a human IgG1 heavy chain according to EU numbering) has been substituted, and in the second heavy chain, at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (in a human IgG1 heavy chain according to EU numbering) has been substituted. In a further embodiment, the first heavy chain and the second heavy chain are substituted at the same positions.In yet a further embodiment, an anti-DR4 antibody is provided, wherein (i) the amino acid at the position corresponding to F405 (according to Eu numbering) is L, or (ii) the amino acid at the position corresponding to K409 (according to Eu numbering) is R. In yet a further embodiment, an anti-DR4 antibody is provided, wherein (i) the amino acid at the position corresponding to F405 (of the human IgG1 heavy chain, according to Eu numbering) is L, or (ii) the amino acid at the position corresponding to K409 (of the human IgG1 heavy chain, according to Eu numbering) is R.

[0229] The present disclosure further provides an anti-DR4 antibody, wherein said anti-DR4 antibody comprises a first heavy chain and a second heavy chain, and wherein one or both heavy chains are modified so as to reduce the extent to which the antibody induces Fc-mediated effector function compared to the same antibody except comprising unmodified first and second heavy chains.

[0230] The anti-DR4 antibody may comprise a first heavy chain and a second heavy chain, wherein in at least one of said first heavy chain and said second heavy chain, one or more amino acids at positions corresponding to positions L234, L235, G236, D265, N297, and P331 in a human IgG1 heavy chain according to Eu numbering are not L, L, G, D, N, and P, respectively. In one embodiment, the anti-DR4 antibody comprises that positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to Eu numbering are F and E, respectively, in said first heavy chain and / or said second heavy chain. In a further embodiment, positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to Eu numbering are F, E, and A, respectively, in said first heavy chain and / or said second heavy chain. In a further embodiment, the positions corresponding to positions L234, L235 and G236 in a human IgG1 heavy chain according to Eu numbering are F, E and R, respectively, in said first heavy chain and / or said second heavy chain.

[0231] The present disclosure further provides an anti-DR4 antibody, wherein the anti-DR4 antibody comprises a first heavy chain and a second heavy chain, wherein the positions in both the first and second heavy chains corresponding to positions L234, L235, and G236 in a human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively, and wherein (i) the position corresponding to F405 in the human IgG1 heavy chain according to Eu numbering is L, or (ii) the position in the first heavy chain corresponding to K409 in the human IgG1 heavy chain according to Eu numbering is R.

[0232] An anti-DR4 antibody in the context of the present invention may comprise a DR4 binding region comprising: (i) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 8 and 9, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, 11 and 12, respectively; (ii) wherein the anti-DR4 antibody comprises a first heavy chain and a second heavy chain, and wherein in both the first heavy chain and the second heavy chain, positions corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively; and (iii) wherein in the first and second heavy chains, the position corresponding to K409 in the human IgG1 heavy chain according to Eu numbering is R, or in the first and second heavy chains, the position corresponding to F405 in the human IgG1 heavy chain according to Eu numbering is L. In one embodiment, the antibody may comprise a DR4 binding region comprising: (i) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 8 and 9, respectively; and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, 11 and 12, respectively; (ii) wherein the anti-DR4 antibody comprises a first heavy chain and a second heavy chain, and wherein in both the first heavy chain and the second heavy chain, positions corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively; and (iii) wherein in the first and second heavy chains, the position corresponding to K409 in the human IgG1 heavy chain according to Eu numbering is R.

[0233] An anti-DR4 antibody in the context of the present invention may comprise the heavy chain sequence shown in SEQ ID NO:19 and the light chain sequence shown in SEQ ID NO:20.

[0234] The present disclosure further provides an anti-DR4 antibody, wherein the EC 50 is in the range of 0.01 to 1.0 μg / mL, for example, in the range of 0.05 to 0.75 μg / mL, for example, in the range of 0.1 to 0.5 μg / mL, when binding to DLD-1, A549, HCT-116, HCT-15, MDA-MB-231 or PANC-1, when assayed, for example, as described in Example 3 herein.

[0235] The present disclosure further provides an anti-DR4 antibody, wherein the EC 50 When monovalently binding to DLD-1, A549, HCT-116, HCT-15, MDA-MB-231 or PANC-1, the antibody has a mAb concentration in the range of 0.1 to 2.0 μg / mL, for example, in the range of 0.25 to 1.75 μg / mL, for example, in the range of 0.4 to 1.75 μg / mL, for example, in the range of 0.5 to 1.5 μg / mL, when assayed, for example, as described in Example 3 herein.

[0236] The present disclosure further provides an anti-DR4 antibody, wherein the EC 50 When bivalently binding to DLD-1, A549, HCT-116, HCT-15, MDA-MB-231 or PANC-1, the antibody has a mAb concentration in the range of 0.1 to 1.0 μg / mL, for example, in the range of 0.15 to 0.75 μg / mL, for example, in the range of 0.2 to 0.5 μg / mL, for example, in the range of 0.25 to 0.4 μg / mL, when assayed, for example, as described in Example 3 herein.

[0237] Antibody production Conventional methods, such as hybrid hybridoma and chemical conjugation methods (Marvin and Zhu (2005) Acta Pharmacol Sin 26:649), can be used to prepare the antibodies of the present invention, including multispecific and bispecific antibodies. The antibodies can be produced by a method comprising (a) culturing a recombinant host cell described herein under conditions in which the antibody is produced, and (b) isolating the antibody produced from the culture. Co-expression in a host cell of two antibodies consisting of different heavy and light chains results in a mixture of potential antibody products in addition to the desired bispecific antibody, which can then be isolated, for example, by affinity chromatography or similar methods.

[0238] As previously described, strategies to support the formation of functional bispecific products by coexpression of different antibody constructs, such as the method described by Lindhofer et al. (1995 J Immunol 155:219), can also be used. Fusion of rat and mouse hybridomas producing different antibodies limits the number of heterodimeric proteins produced by preferential species-restricted heavy / light chain pairing. Another strategy to promote heterodimer formation over homodimer formation is the "knob-into-hole" strategy, in which a protrusion on the first heavy chain polypeptide and a corresponding cavity in the second heavy chain polypeptide are introduced, so that the protrusion can enter the cavity at the interface of these two heavy chains, promoting heterodimer formation and preventing homodimer formation. The "protrusion" is constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains. A compensatory "cavity" of the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (U.S. Pat. No. 5,731,168). European Patent No. 1870459 (Chugai) and International Publication No. 2009089004 (Amgen) describe other strategies for supporting heterodimer formation by co-expressing different antibody domains in host cells. In these methods, one or more residues constituting the CH3-CH3 interface of both CH3 domains are replaced with charged amino acids to electrostatically disfavor homodimer formation and favor heterodimerization. International Publication No. 2007110205 (Merck) describes yet another strategy that exploits the differences between IgA and IgG CH3 domains to promote heterodimerization.

[0239] Another in vitro method for producing bispecific antibodies is described in WO2008119353 (Genmab), in which the bispecific antibodies are formed by "Fab arm" or "half molecule" exchange (exchange of heavy chains and binding light chains) between two monospecific IgG4 or IgG4-like antibodies by incubation under reducing conditions. The resulting product is a bispecific antibody with two Fab arms that may contain different sequences.

[0240] Preferred methods for preparing the bispecific FAPαxDR4 antibodies of the present invention include those described in WO2011131746 and WO13060867 (Genmab), which comprise the steps of: a) providing a first antibody comprising an Fc region, the Fc region comprising a first CH3 region; b) providing a second antibody comprising a second Fc region, the Fc region comprising a second CH3 region; wherein the first antibody is a FAPα antibody and the second antibody is a DR4 antibody, or vice versa; where: the sequences of the first and second CH3 regions are different, whereby a heterodimeric interaction between the first and second CH3 regions is stronger than each of the homodimeric interactions of the first and second CH3 regions; c) incubating the first antibody with the second antibody under reducing conditions; and d) obtaining said bispecific FAPαxDR4 antibody; Process.

[0241] Likewise, the present invention relates to a method for producing a multispecific, such as a bispecific, antibody according to the invention, said method comprising the steps of: a) providing a first homodimeric antibody comprising a FAPα-binding region as described herein, and a second homodimeric antibody comprising a DR4-binding region as described herein, said antibodies comprising an Fc region and optionally containing further features as described herein; wherein the sequences of the first CH3 region and the second CH3 region of the first antibody and the second antibody are different, whereby a heterodimeric interaction between the first and second CH3 regions is stronger than a homodimeric interaction of the first CH3 region and a homodimeric interaction of the second CH3 region, respectively; b) incubating the first antibody with the second antibody under reducing conditions sufficient to allow cysteines in the hinge region to undergo disulfide bond isomerization; and c) obtaining a heterodimeric multispecific antibody of the invention as described herein, comprising a first immunoglobulin heavy chain and a first immunoglobulin light chain of the first antibody, and a second immunoglobulin heavy chain and a second immunoglobulin light chain of the second antibody; This includes:

[0242] In one embodiment, the first antibody is incubated with the second antibody under reducing conditions sufficient to allow cysteines in the hinge region to undergo disulfide bond isomerization, and wherein the heterodimeric interaction between the first and second antibodies in the resulting heterodimeric antibody is such that no Fab arm exchange occurs at 37° C. with 0.5 mM GSH after 24 hours.

[0243] Without being limited by theory, in step c), heavy chain disulfide bonds in the hinge region of the parent antibody are reduced, and the resulting cysteines can then form inter-heavy chain disulfide bonds with cysteine ​​residues of another parent antibody molecule (originally with a different specificity). In one embodiment of this method, the reducing conditions of step c) include the addition of a reducing agent, e.g., a reducing agent selected from the group consisting of 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and beta-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. In a preferred embodiment, the reducing agent is 2-mercaptoethylamine. In a further embodiment, step c) includes restoring the conditions to non-reducing or less reducing, e.g., by removing the reducing agent, e.g., by desalting.

[0244] In a further aspect, the present invention relates to a method for producing a multispecific antibody, said method comprising: (a)(i) a first antibody that is a monospecific anti-FAPα antibody described herein; and a second antibody comprising a DR4 binding region comprising a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 8 and 9, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, 11 and 12, respectively; a first and a second antibody, wherein the antibodies comprise a first heavy chain and a second heavy chain, and the positions in both the first heavy chain and the second heavy chain that correspond to positions L234, L235, and G236 in a human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively; (ii) a second antibody that is a monospecific anti-DR4 antibody described herein, and A first antibody comprising a FAPα-binding region comprising a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 1, 2 and 3, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 4, 5 and 6, respectively, a first and a second antibody, wherein the antibodies comprise a first heavy chain and a second heavy chain, and wherein the positions in both the first heavy chain and the second heavy chain that correspond to positions L234, L235, and G236 in a human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively; or (iii) a first antibody that is a monospecific anti-FAPα antibody described herein and a second antibody that is a monospecific anti-DR4 antibody described herein; Provide; wherein the sequences of the first and second CH3 regions of the first and second antibodies are different, such that the heterodimeric interaction between the first and second CH3 regions is stronger than the homodimeric interaction of the first CH3 region and the homodimeric interaction of the second CH3 region, respectively, and preferably, the amino acid at the position corresponding to F405 in the first CH3 region is L and the amino acid at the position corresponding to K409 in the second CH3 region is R, or vice versa; (b) incubating the first antibody with the second antibody under reducing conditions sufficient to allow cysteines in the hinge region to undergo disulfide bond isomerization; and (c) obtaining a multispecific antibody comprising the first immunoglobulin heavy chain and the first immunoglobulin light chain of the first antibody, and the second immunoglobulin heavy chain and the second immunoglobulin light chain of the second antibody;

[0245] In a further aspect, the present invention relates to a method for producing a multispecific antibody, said method comprising: (a)(i) a first antibody that is a monospecific anti-FAPα antibody described herein; and a second antibody comprising a DR4 binding region, wherein the heavy chain variable region (VH) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 8 and 9, respectively, and the light chain variable region (VL) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, 11 and 12, respectively; a first and a second antibody, wherein the antibodies comprise a first heavy chain and a second heavy chain, and the positions in both the first heavy chain and the second heavy chain that correspond to positions L234, L235, and G236 in a human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively; (ii) a second antibody that is a monospecific anti-DR4 antibody described herein, and A first antibody comprising a FAPα-binding region, wherein the heavy chain variable region (VH) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 1, 2 and 3, respectively, and the light chain variable region (VL) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 4, 5 and 6, respectively; a first and a second antibody, wherein the antibodies comprise a first heavy chain and a second heavy chain, and wherein the positions in both the first heavy chain and the second heavy chain that correspond to positions L234, L235, and G236 in a human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively; or (iii) a first antibody that is a monospecific anti-FAPα antibody described herein and a second antibody that is a monospecific anti-DR4 antibody described herein; wherein the sequences of the first and second CH3 regions of the first and second antibodies are different, such that the heterodimeric interaction between the first and second CH3 regions is stronger than each of the homodimeric interactions of the first and second CH3 regions, and preferably, the amino acid at the position corresponding to F405 in the first CH3 region is L, and the amino acid at the position corresponding to K409 in the second CH3 region is R; (b) incubating the first antibody with the second antibody under reducing conditions sufficient to allow cysteines in the hinge region to undergo disulfide bond isomerization; and (c) obtaining a multispecific antibody comprising the first immunoglobulin heavy chain and the first immunoglobulin light chain of the first antibody, and the second immunoglobulin heavy chain and the second immunoglobulin light chain of the second antibody;

[0246] In a further aspect, the present invention relates to a method comprising the steps of: (a) culturing a host cell containing an expression vector comprising (i) a nucleic acid sequence encoding a heavy chain sequence of a FAPα-binding region defined herein, and (ii) a nucleic acid sequence encoding a light chain sequence of a FAPα-binding region defined herein, and purifying the first antibody from the culture medium; (b) culturing a host cell containing an expression vector comprising (iii) a nucleic acid sequence encoding a heavy chain sequence of a DR4 binding region as defined herein, and (iv) a nucleic acid sequence encoding a light chain sequence of a DR4 binding region as defined herein, and purifying the second antibody from the culture medium; (c) incubating the first antibody with the second antibody under reducing conditions sufficient to allow cysteines in the hinge region to undergo disulfide bond isomerization, thereby obtaining a bispecific antibody.

[0247] In one embodiment, step c) comprises the addition of a reducing agent. In a further embodiment, step c) comprises the addition of a reducing agent selected from the group consisting of 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and beta-mercaptoethanol. In yet a further embodiment, step c) comprises the addition of a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. In yet a further embodiment, step c) comprises restoring conditions to non-reducing or less reducing, for example, by removal of the reducing agent. In a further embodiment, the reducing agent is removed by desalting.

[0248] In a further aspect, the present invention relates to a multispecific antibody obtained by the method described herein.

[0249] Anti-idiotype antibodies In a further aspect, the present invention relates to an antibody comprising at least one antigen-binding region capable of binding to DR4 and / or FAPα, i.e. an anti-idiotypic antibody that binds to an antibody according to the invention described herein. In a particular embodiment, the anti-idiotypic antibody binds to an antigen-binding region capable of binding to DR4 and / or FAPα.

[0250] Anti-idiotypic (Id) antibodies are antibodies that recognize unique determinants typically associated with the antigen-binding site of an antibody. Anti-Id antibodies can be prepared by immunizing an animal of the same species and genetic type as the source of the monoclonal antibody with the monoclonal antibody from which the anti-Id is prepared. The immunized animal is typically able to recognize and respond to the idiotypic determinants of the immunizing antibody by producing antibodies against these idiotypic determinants (anti-Id antibodies). Such antibodies are described, for example, in U.S. Pat. No. 4,699,880. Such antibodies are a further feature of the present invention.

[0251] An anti-Id antibody can also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody. An anti-anti-Id antibody can be epitopically identical to the original monoclonal antibody that induced the anti-Id antibody. Thus, by using antibodies against the idiotypic determinants of a monoclonal antibody, it is possible to identify other clones expressing antibodies of the same specificity. The anti-Id antibody can be modified (thereby producing anti-Id antibody variants) and / or derivatized by any suitable technique, such as those described elsewhere herein for the DR4-specific and / or FAPα-specific antibodies of the present invention. For example, a monoclonal anti-Id antibody can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize BALB / c mice. Serum from these mice typically contains anti-anti-Id antibodies with binding characteristics similar, if not identical, to those of the original / parent antibody. [Table 3] TIFF2025530208000004.tif234154TIFF2025530208000005.tif231153TIFF2025530208000006.tif220154 TIFF2025530208000007.tif219153TIFF2025530208000008.tif232153TIFF2025530208000009.tif223153 TIFF2025530208000010.tif232153TIFF2025530208000011.tif232154TIFF2025530208000012.tif219153 TIFF2025530208000013.tif232154TIFF2025530208000014.tif232154TIFF2025530208000015.tif219153 TIFF2025530208000016.tif231155TIFF2025530208000017.tif233155TIFF2025530208000018.tif232153 TIFF2025530208000019.tif233153TIFF2025530208000020.tif233154TIFF2025530208000021.tif232154 TIFF2025530208000022.tif234155TIFF2025530208000023.tif235154TIFF2025530208000024.tif232153 TIFF2025530208000025.tif232154TIFF2025530208000026.tif197154TIFF2025530208000027.tif173154

[0252] Further items of this disclosure This invention is further illustrated by the following examples which should not be construed as further limiting.

[0253] [Example] [Example 1] Antibody generation a.FAPα antibody Immunization and hybridoma generation were performed at Aldevron GmbH (Freiburg, Germany). The construct used for immunization was a cDNA encoding full-length human FAPα (SEQ ID NO: 33) cloned into Aldevron's proprietary immunization vector. Anti-FAPα antibodies were generated by gene (DNA) immunization of OmniRat animals (transgenic rats expressing a diverse repertoire of antibodies with fully human idiotypes; Ligand Pharmaceuticals Inc.) using a gene gun. Serum samples were collected after a series of immunizations and tested by flow cytometry using mammalian cells transiently transfected with an expression plasmid stably expressing human FAPα (SEQ ID NO: 33). Antibody-producing cells were isolated from rat spleens and fused with mouse myeloma cells (Ag8) according to standard procedures. RNA from hybridomas producing FAPα-specific antibodies was extracted for sequencing. The heavy and light chain variable regions (VH and VL domains) of interest were gene synthesized and cloned into expression vectors containing the framework sequences of the human IgG1 heavy chain constant region (SEQ ID NO: 21) or the human kappa light chain constant region (LC) (SEQ ID NO: 27) of the human IgG1m(f) allotype, depending on the binding domain selected. The FAPα-specific IgG1 kappa antibody IgG1-FAPα was selected with the variable domain sequences of SEQ ID NO: 14 (VL) and SEQ ID NO: 13 (VH).

[0254] Other FAPα-targeting antibodies used in the examples: FAP5, disclosed in US Patent Application Publication No. 20090304718A1, ESC11, disclosed in WO 2011040972A1, and RG7386, disclosed in US Patent Application Publication No. 9926379B2. Antibodies were generated according to standard procedures (described in sections d-f).

[0255] b.DR4 antibody Immunization and hybridoma generation were performed at Aldevron GmbH (Freiburg, Germany). The constructs used for immunization were a cDNA encoding full-length human DR4 (SEQ ID NO: 68) cloned into an Aldevron-proprietary immunization vector, and a cDNA encoding the extracellular domain (ECD) of human DR4 (aa 24-239 of SEQ ID NO: 68) cloned into an Aldevron-proprietary immunization vector with a vector-derived N-terminal tag sequence. Anti-DR4 antibodies were generated by genetic (DNA) immunization of OmniRat animals using a gene gun. Serum samples were collected after a series of immunizations and tested by flow cytometry using mammalian cells transiently transfected with an expression plasmid for human DR4 expression. Antibody-producing cells were isolated from rat spleens and fused with mouse myeloma cells (Sp2.0) according to standard procedures. RNA from hybridomas producing DR4-specific antibodies was extracted for sequencing. The heavy and light chain variable regions (VH and VL domains) of interest were gene synthesized and cloned into expression vectors containing the framework sequences of the human IgG1 heavy chain constant region (SEQ ID NO: 21) of the human IgG1m(f) allotype or the human lambda light chain constant region (LC) (SEQ ID NO: 28), depending on the selected binding domain. The DR4-specific IgG1 lambda antibody IgG1-DR4 was selected with the variable domain sequences of SEQ ID NO: 16 (VL) and SEQ ID NO: 15 (VH).

[0256] Other DR4-targeting antibodies used in the experiments: T1014A04, disclosed in U.S. Patent No. 7,361,341 B2, chCTB007, disclosed in U.S. Patent Application Publication No. 20090136503 A1, and ABBV-621, a TRAIL-R fusion agonist, disclosed in WO 2019178438 A1. Antibodies were generated according to standard procedures (described in sections d-f).

[0257] c. Control antibody A human IgG1 antibody with an antigen-binding domain identical to that of the HIV-1 gp120-specific antibody b12 was used as a negative, non-binding control in some experiments (Barbas et al., J Mol Biol. 1993 Apr 5;230(3):812-2). The VH and VL domains of b12 were generated by de novo gene synthesis (GeneArt Gene Synthesis; ThermoFisher Scientific, Germany) and cloned into an expression vector containing the framework sequence of the human IgG1 heavy chain constant region of the human IgG1m(f) allotype. The sequences of the heavy and light chains of the b12 control antibody are included in section g below.

[0258] d. Antibody expression Antibodies were obtained by transfection of heavy and light chain expression vectors into production cell lines and purified from culture supernatants by protein A affinity chromatography for functional characterization. IgG concentrations were measured by absorbance at 280 nm. Purified antibodies were stored in phosphate-buffered saline (PBS) at 4°C.

[0259] e. Generation of bispecific antibodies Bispecific antibodies were obtained by controlled Fab arm exchange (DuoBody® platform technology), i.e., 2-MEA-induced controlled Fab arm exchange (cFAE) as described in WO 2011147986, WO 2011131746 and WO 2013060867 (Genmab), and by Labrijn et al. (Labrijn et al., PNAS 2013, 110:5145-50; Gramer et al., Mabs 2013, 5:962-973). Briefly, two parent antibodies containing single, matched point mutations in the CH3 domain (one F405L and the other K409R [Eu numbering (Kabat, NIH publication no. 91-3242, 5th edition ed. National Institutes of Public Health, Bethesda, MD, USA. 662, 680, 689)]) were generated separately, mixed, and subjected to controlled reduction conditions. The reduction conditions disrupt the interchain disulfide bonds of the molecules, while the matched CH3 domains (containing F405L and K409R) drive heterodimerization of the Fab arms and the formation of bispecific molecules. Subsequent reoxidation of the disulfide bonds yields highly pure bispecific antibody preparations with regular IgG1 structure.

[0260] f.Fc mutation To minimize interaction with Fcγ receptors and the complement component C1q, the mutations L234F, L235E, and D265A (FEA; Engelberts et al., EBiomedicine, 2020; SEQ ID NO: 23) or L234F, L235E, and G236R (FER, WO 2022 / 189667, SEQ ID NO: 22) according to EU numbering were introduced into the heavy chain constant domain.

[0261] Antibodies with both FEA and F405L or K409R mutations introduced are referred to as FEAL or FEAR, respectively, in the experiments shown. Antibodies with both FER and F405L or K409R mutations introduced are referred to as FERL or FERR, respectively, in the experiments shown.

[0262] To generate bispecific antibodies, mix two parent antibodies in PBS buffer (phosphate-buffered saline; 8.7 mM HPO4 2- , 1.8mM H2PO4 - , 163.9mM Na + , 140.3 mM Cl - The antibodies were mixed in equal volumes in PBS (pH 7.4). 2-Mercaptoethylamine-HCl (2-MEA) was added to a final concentration of 75 mM, and the reaction mixture was incubated at 31°C for 5 hours. To reoxidize the interchain disulfide bonds and allow the formation of intact bispecific antibodies, 2-MEA was removed by dialysis into PBS buffer using a 10 kDa molecular weight cutoff Slide-A-Lyzer support (Thermo Fisher Scientific) according to the manufacturer's protocol.

[0263] g. Overview of antibodies used in the examples The amino acid sequences of the antibodies used in the following experiments, for example the parent antibodies of the bispecific and / or monospecific antibodies, are shown in the following SEQ ID NOs:

[0264] IgG1-FAPα-FERL: SEQ ID NO: 17 (HC) and SEQ ID NO: 18 (LC) IgG1-FAPα-FEAL: SEQ ID NO: 29 (HC) and SEQ ID NO: 18 (LC) IgG1-DR4-FERR: SEQ ID NO: 19 (HC) and SEQ ID NO: 20 (LC) IgG1-DR4-FEAR: SEQ ID NO: 30 (HC) and SEQ ID NO: 20 (LC) IgG1-DR4-FEAL: SEQ ID NO: 32 (HC) and SEQ ID NO: 20 (LC) IgG1-b12-FERL: SEQ ID NO: 60 (HC) and SEQ ID NO: 57 (LC) IgG1-b12-FEAL: SEQ ID NO: 58 (HC) and SEQ ID NO: 57 (LC) IgG1-b12-FERR: SEQ ID NO: 61 (HC) and SEQ ID NO: 57 (LC) IgG1-b12-FEAR: SEQ ID NO: 59 (HC) and SEQ ID NO: 57 (LC) IgG1-b12: SEQ ID NO: 56 (HC) and SEQ ID NO: 57 (LC) IgG1-FAP5-FEAL: SEQ ID NO: 47 (HC) and SEQ ID NO: 48 (LC) IgG1-FAP5: SEQ ID NO: 49 (HC) and SEQ ID NO: 48 (LC) IgG1-FAPα-F405L: SEQ ID NO: 31 (HC) and SEQ ID NO: 18 (LC) IgG1-FAP-ESC11-F405L: SEQ ID NO: 50 (HC) and SEQ ID NO: 51 (LC) IgG1-DR4-T1014A04-FEAR: SEQ ID NO: 52 (HC) and SEQ ID NO: 53 (LC) IgG1-DR4-chCTB007-FEAR: SEQ ID NO: 54 (HC) and SEQ ID NO: 55 (LC) ABBV-621-Fc fusion: SEQ ID NO: 65 RG7386: SEQ ID NO: 62 (HC) and SEQ ID NOs: 63-64 (LC) IgG1-b12-FER: SEQ ID NO: 73 (HC) and SEQ ID NO: 57 (LC)

[0265] [Example 2] Binding to human lung fibroblasts and CAFs a. Binding of BisG1-FAPα-FERL / DR4-FERR to FAPα expressed on the cell surface. The binding of bispecific or monoclonal antibodies (BisG1-FAPα-FERL / DR4-FERR, BisG1-FAPα-FERL / b12-FERR, and IgG1-FAPα-FERL) with an anti-FAPα arm derived from IgG1-FAPα-FERL to cell surface-expressed human FAPα was analyzed by flow cytometry using human lung fibroblasts and CAFs that endogenously express FAPα. BisG1-b12-FERL / b12-FERL was used as a negative control.

[0266] Cell viability of human lung fibroblasts (Coriell Institute, catalog no. GM05389) and CAFs (grown from primary human CRC biopsies; Strating et al., Front. Immunol. 2023, 16:14:1053920) was measured using acridine orange / propidium iodide (AO / PI; Nexcelom, catalog no. CS2-0106). Human lung fibroblasts and CAFs (50,000 cells / well) were seeded into 96-well round-bottom plates (Greiner Bio-one, catalog no. 650101). Antibody dilutions were prepared using fluorescence-activated cell sorting (FACS) buffer consisting of phosphate-buffered saline (PBS, Lonza, catalog no. BE17-517Q) + 1% bovine serum albumin (BSA, Roche, catalog no. 10735086001) + 0.02% sodium azide (Bio-World, catalog no. 41920044-3). The plate was centrifuged, the supernatant removed, and the cells resuspended in 50 μL of human Fc Block (BD, catalog no. 564220, diluted 1:100 in FACS buffer) and 50 μL of viability stain TO-PRO-3 Iodide (Thermo Fisher, catalog no. T3605, diluted 1:25,000 in FACS buffer) and incubated for 15 min at 4°C. The plate was washed three times with FACS buffer. The plates were centrifuged, the supernatant removed, and the cells were resuspended in 50 μL of antibody dilutions (concentrations ranging from 90 to 0.0005 μg / mL, 3-fold serial dilutions in FACS buffer) of BisG1-FAPα-FERL / DR4-FERR, BisG1-FAPα-FERL / b12-FERR, BisG1-b12-FERL / b12-FERR (for CAF cultures only, the top six concentrations), or IgG1-FAPα-FERL (for CAF cultures only, the top four concentrations) and incubated for 30 min at 4 °C. The cells were washed three times with FACS buffer and resuspended in 50 μL of fluorescein isothiocyanate (FITC)-labeled polyclonal antibody goat anti-mouse IgG1 (Jackson Immuno Research, catalog number 109-096-097, diluted 1:100 in FACS buffer).After 30 minutes of incubation at 4°C, cells were washed twice with FACS buffer and resuspended in FACS buffer. Antibody binding to viable cells (TO-PRO-3 negative) was analyzed by flow cytometry on a FACSCelesta™ (BD biosciences), and data were processed using FlowJo_v10.8.1 (FlowJo LLC). Geometric mean fluorescence intensity (gMFI) was determined and visualized using GraphPad Prism. Binding curves were analyzed using nonlinear regression analysis. 50% of maximal effect (EC 50 The concentrations (μg / mL) at which the observed α- and β-blocking effects were observed were derived from the fitted curves.

[0267] result Similar dose-dependent human FAPα binding profiles were observed for both bispecific antibodies containing a FAPα-specific arm (i.e., BisG1-FAPα-FERL / DR4-FERR and BisG1-FAPα-FERL / b12-FERR) on human lung fibroblasts and CAFs (EC values ​​from three experiments). 50 (See Figures 1A-1B and Table 1). Lower maximal binding was observed for the bivalent monoclonal antibody IgG1-FAPα-FERL. No binding was observed for the negative control BisG1-b12-FERL / b12-FERR antibody.

[0268] [Table 4]

[0269] b. Superior monovalent binding of FAPα clones compared to FAP5 clones The binding of FAPα-targeting monoclonal antibody clones, IgG1-FAPα-FEAL and IgG1-FAP5-FEAL, and bispecific antibodies (BisG1-FAPα-FEAL / b12-FEAR and BisG1-FAP5-FEAL / b12-FEAR) with binding arms derived from two FAPα-binding clones to human lung fibroblasts that endogenously express FAPα was compared and analyzed by flow cytometry. IgG1-b12-FEAR was used as a negative control.

[0270] Binding assays and analyses were performed as detailed in section a with the following differences: 20,000 fibroblasts / well were seeded, no viability staining was performed, and primary antibody dilutions were tested (10–0.0001 μg / mL final concentrations, serial 5-fold dilutions using FACS buffer).

[0271] result All antibodies containing a FAPα-binding Fab arm showed dose-dependent binding to human FAPα in human lung fibroblasts (Figure 1C and Table 2). The apparent affinities (EC 50 ) was superior to that containing the FAPα-binding domain from the FAP5 clone (lower EC 50 Furthermore, in contrast to FAP5, lower maximal binding was observed for the bivalent antibody IgG1-FAPα-FEAL compared to the monovalent antibody BisG1-FAPα-FEAL / b12-FEAR, indicating that binding was more similar in the monovalent format compared to the bivalent format. No binding was observed for the negative control IgG1-b12-FEAR.

[0272] [Table 5]

[0273] [Example 3] Binding of DR4-specific antibodies to cell surface-expressed DR4 a. Efficient monovalent and bivalent binding of anti-DR4 antibodies to multiple DR4-expressing tumor cell lines. The binding of BisG1-FAPα-FERL / DR4-FERR, BisG1-b12-FERL / DR4-FERR, and IgG1-DR4-FERR to cell surface-expressed human DR4 was analyzed by flow cytometry using six human cancer cell lines that endogenously express DR4: colorectal adenocarcinoma (DLD-1, HCT-15), non-small cell lung carcinoma (A549), colon carcinoma (HCT-116), triple-negative breast carcinoma (MDA-MB-231), and pancreatic ductal adenocarcinoma (PANC-1). IgG-b12 was included as a negative control.

[0274] Cancer cell lines (DLD-1: ATCC, Catalog No. CCL-221; HCT-15: ATCC, Catalog No. CCL-225; HCT-116: ATCC, Catalog No. CCL-247; A549: ATCC, Catalog No. CCL-185; MDA-MB-231: ATCC, Catalog No. HTB-26; PANC-1: ATCC, Catalog No. CRL-1469) were detached from cell culture flasks using trypsin solution (Gibco, Catalog No. 25300-054), washed in PBS (GE Healthcare, Catalog No. SH3A3830.03), counted, and resuspended at the desired concentration.

[0275] Binding assays were performed as detailed in Example 2, section a, except for the use of IgG1-b12 antibody, and only the top three concentrations were tested. Data were processed using FlowJo_v10.8.1. gMFI was determined and visualized using GraphPad Prism.

[0276] result The DR4-specific antibody exhibited dose-dependent binding to all human DR4-expressing cancer cell lines (Figures 2A-F). Average EC values ​​from three independent experiments 50The values ​​are shown in Table 3. As observed for binding to FAPα, the maximum binding capacity for all cell lines was higher for the monovalent antibody variants compared to the bivalent antibody variants. The bivalent IgG1-DR4-FERR antibody had a lower EC compared to the monovalent antibody BisG1-FAPα-FERL / DR4-FERR for all cell lines. 50 Strong binding of both monovalent and bivalent binding formats to DR4 is observed.

[0277] BisG1-b12-FERL / DR4-FERR exhibited similar binding and mean EC 50 whereas the negative control antibody IgG1-b12 showed no binding, confirming that the observed binding was dependent on the DR4-specific arm.

[0278] [Table 6]

[0279] b. Monovalent binding of chCTB007 and T1014A04 antibodies to cell surface-expressed DR4 is reduced compared to bivalent binding. Binding of the DR4-specific antibodies BisG1-b12-FEAL / DR4-T1014A04-FEAR, BisG1-b12-FEAL / DR4-chCTB007-FEAR, IgG1-DR4-chCTB007-FEAR, and IgG1-DR4-T1014A04-FEAR to cell surface-expressed human DR4 was analyzed by flow cytometry using the human cancer cell line OPM-2 (DSMZ, catalog no. ACC 50), which endogenously expresses DR4. IgG1-b12 was used as a negative control.

[0280] Binding assays were performed as detailed in Example 2, section a, with the following modifications: no viability stain or Fc Block was added; an antibody concentration series (10 to 0.0001 μg / mL final concentrations, serial 4-fold dilutions in FACS buffer); secondary antibody: R-phycoerythrin (PE)-labeled polyclonal antibody goat anti-human IgG1 (Jackson Immuno Research, catalog number 109-116-098, 1:500 dilution in FACS buffer); and a full concentration curve of a negative control antibody was included using an iQue Plus flow cytometer.

[0281] Furthermore, the binding of the DR4-specific antibodies IgG1-DR4-FERR, BisG1-b12-FERL / DR4-FERR, BisG1-FAPα-FERL / DR4-FERR, IgG1-DR4-chCTB007-FEAR, BisG1-b12-FEAL / DR4-chCTB007-FEAR, IgG1-DR4-T1014A04-FEAR, and BisG1-b12-FEAL / DR4-T1014A04-FEAR to cell surface-expressed human DR4 was analyzed by flow cytometry using the human carcinoma cell line MDA-MB-231, which endogenously expresses DR4. BisG1-b12-FERL / b12-FERR was used as a negative control.

[0282] Binding assays were performed as detailed in Example 2, section a, with the following modification: a full concentration curve of a negative control antibody was included.

[0283] result IgG1-DR4-T1014A04-FEAR and IgG1-DR4-chCTB007-FEAR showed dose-dependent binding to human DR4 expressed by the human tumor cell line OPM-2 (Figure 2G). Reduced binding of IgG1-DR4-T1014A04-FEAR was observed compared to IgG1-DR4-chCTB007. Furthermore, low binding of the monovalent binding variant BisG1-b12-FEAL / DR4-chCTB007-FEAR was observed, while no binding or low binding of the monovalent binding variant BisG1-b12-FEAL / DR4-T1014A04-FEAR was detected.

[0284] IgG1-DR4-FERR showed dose-dependent binding to human DR4 expressed by the human tumor cell line MDA-MB-231 with apparent affinity and maximum MFI similar to those of IgG1-DR4-chCTB007-FEAR, but with higher apparent affinity and maximum MFI compared to IgG1-DR4-T1014A04-FEAR. The monovalent binding of BisG1-b12-FERL / DR4-FERR and BisG1-FAPα-FERL / DR4-FERR to DR4 expressed by MDA-MB-231 cells was stronger (higher apparent affinity and maximum MFI) compared to BisG1-b12-FEAL / DR4-chCTB007-FEAR and BisG1-b12-FEAL / DR4-T1014A04-FEAR (Figure 2H, Table 4).

[0285] [Table 7]

[0286] [Example 4] Species cross-reactivity to FAPα orthologues The binding of BisG1-FAPα-FERL / DR4-FERR, BisG1-FAPα-FEAL / b12-FEAR, and IgG1-FAPα-FERL to FAPα from species commonly used in nonclinical toxicology studies was assessed by flow cytometry using Expi293F cells transiently expressing FAPα from various animal species. IgG1-b12 was included as a negative control.

[0287] ExpiFectamine™ 293 transfection reagent (Thermo Fisher Scientific, Cat. No. A14525), Opti-MEM™ reduced serum medium, GlutaMAX™ supplement (Thermo Fisher Scientific, Cat. No. 51985026), and transfection enhancers 1 and 2 (Thermo Fisher Scientific, Cat. No. A14525) were used according to the manufacturer's instructions to transfect Expi293F suspension cells (Thermo Fisher Scientific, Cat. No. A14527) with full-length human (UniProt ID Q12884, SEQ ID NO: 33), cynomolgus monkey (Macaca fascicularis, UniProt ID A0A2K5VGF4, SEQ ID NO: 39), dog (Canis familiaris, UniProt ID A0A8C0NKP1, SEQ ID NO: 37), pig (Sus scrofa, UniProt ID K7GQN2, SEQ ID NO: 38), rat (Rattus norvegicus, UniProt ID A0A8C0NKP1, SEQ ID NO: 39), and guinea pig (Guinea pig, UniProt ID A0A8C0NKP1, SEQ ID NO: 39). The mice were transiently transfected with the mammalian expression vector pSB encoding the FAPα orthologues of either mouse (Mus musculus, UniProt ID P97321, SEQ ID NO: 35) or mouse (Mus musculus, UniProt ID P97321, SEQ ID NO: 36) or mouse (Mus musculus, UniProt ID P97321, SEQ ID NO: 35) FAPα orthologues.

[0288] Expi293F cells expressing recombinant FAPα from various species (human, cynomolgus monkey, dog, pig, rat, or mouse) were seeded (20,000 cells / well) in 96-well round-bottom plates (Greiner Bio-one, catalog no. 650101). Antibody dilutions were prepared using FACS buffer consisting of PBS (Capricorn Scientific, catalog no. PBS-10XA, diluted to 1X PBS in distilled water) + 1% BSA (Roche, catalog no. 10735086001) + 0.02% sodium azide (Bio-World, catalog no. 41920044-3). The plates were centrifuged, the supernatant removed, and the cells were resuspended in 100 μL of antibody dilutions of BisG1-FAPα-FERL / DR4-FERR, BisG1-FAPα-FEAL / b12-FEAR, IgG1-FAPα-FERL, and IgG1-b12 (concentrations ranging from 30 to 0.00017 μg / mL, 3-fold serially diluted in FACS buffer) and incubated at 4 °C for 30 min. The cells were washed with FACS buffer and resuspended in 50 μL of PE-conjugated goat anti-human IgG (Jackson ImmunoResearch, catalog number 106-116-098; diluted 1:400 in FACS buffer). After a 30-min incubation at 4 °C, the cells were washed with FACS buffer and resuspended in FACS buffer supplemented with TO-PRO-3 iodide viability marker (Invitrogen, catalog number T3605, diluted 1:4,000). Antibody binding to viable cells (gMFI of PE on TO-PRO-3-negative cells) was analyzed by flow cytometry with FACS Celesta and FACS_Diva software (first two experiments) or iQue®3 and FlowJo software (last experiment). gMFI was determined and visualized using GraphPad Prism. Binding curves were analyzed using nonlinear regression analysis. 50% of maximal effect (EC 50 The concentrations (μg / mL) at which the observed α- and β-blocking effects were observed were derived from the fitted curves.

[0289] result BisG1-FAPα-FERL / DR4-FERR showed dose-dependent binding to all species tested (Figures 3A-F). The apparent affinities for human and cynomolgus monkey FAPα were similar (Figures 3A-B), with mean EC 50 The EC values ​​were 0.24 ± 0.04 μg / mL and 0.25 ± 0.055 μg / mL, respectively (Table 5). All antibodies tested showed reduced binding to FAPα from mouse, rat, dog, and pig compared to human (Figures 3C-3F), suggesting that the EC values ​​for these species are not significant. 50 could not be calculated.

[0290] BisG1-FAPα-FEAL / b12-FEAR and BisG1-FAPα-FERL / DR4-FERR showed similar dose-dependent binding profiles, indicating that binding was FAPα-specific. Bivalent binding with the IgG1-FAPα-FERL antibody demonstrated higher binding than monovalent binding in most species, except for humans (similar binding) and cynomolgus monkeys (lower maximum binding). No binding of BisG1-FAPα-FERL / DR4-FERR to untransfected control cells was observed (data not shown), nor was IgG1-b12 binding to FAPα in any of the species tested (Figure 3A-F).

[0291] In summary, BisG1-FAPα-FERL / DR4-FERR and IgG1-FAPα-FERL showed cross-reactivity with all species tested, with highest and comparable binding to human and cynomolgus monkey FAPα.

[0292] [Table 8]

[0293] [Example 5] Binding affinity of FAPα clones to recombinant human and cynomolgus monkey FAPα. The binding affinities of BisG1-FAPα-FERL / DR4-FERR, BisG1-FAPα-FERL / b12-FERR, and control BisG1-b12-FERL / DR4-FERR to recombinant human and cynomolgus monkey FAPα proteins were determined using label-free biolayer interferometry on an Octet HTX instrument (Sartorius).

[0294] Experiments were performed at 30°C with shaking at 1,000 RPM. Amine-reactive second-generation (AR2G) biosensors (Sartorius, catalog no. 18-5092) were activated by reacting with 20 mM EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) (Sigma-Aldrich, catalog no. 03449) and 10 mM s-NHS (N-hydroxysulfosuccinimide sodium salt) (Sigma-Aldrich, catalog no. 56485) for 300 seconds. The activated AR2G sensors were loaded with 2.5 μg / mL His-tagged recombinant human FAPα (Acro Biosystems, Cat. No. FAP-H5244-100ug) in 10 mM sodium acetate pH 5.0 (Sartorius, Cat. No. 18-1069) or 2.5 μg / mL His-tagged recombinant cynomolgus monkey FAPα (Acro Biosystems, Cat. No. FAP-C52H3-100ug) in 10 mM sodium acetate pH 5.0 for 600 seconds and quenched with 1 M ethanolamine pH 8.5 (Sartorius Cat. No. 18-1071) for 300 seconds. After a baseline measurement in Sample Diluent (1000 seconds; Sartorius, catalog no. 18-1104), the association (100 seconds) and dissociation (4000 seconds) of functional monovalent antibodies were determined using a concentration range of 0.78 nM to 800 nM in two-fold serial dilutions in Sample Diluent. The molecular weights of the antibodies used in the calculations were calculated from their sequences.

[0295] Data were acquired using Data Acquisition Software v12 (Sartorius) and analyzed with Data Analysis Software v12 (Sartorius). Antibody data traces were corrected by subtracting a reference sensor incubated with Sample Diluent instead of antibody.

[0296] The Y-axis was aligned to the last 10 seconds of baseline. Interstep Correction alignment for dissociation and Savitzky-Golay filtering were applied. Data traces with a response <0.05 nM were excluded from analysis. K lower than 50 nM D For antibodies with values, data traces with concentrations above 100 nM were also excluded. Data were fitted with a 1:1 global full-fit model using a 100 s association and dissociation time set with a window of interest of 4,000 s.

[0297] result BisG1-FAPα-FERL / DR4-FERR has comparable picomolar K D The values ​​are shown in Table 6. The bispecific antibody with the non-binding control arm BisG1-FAPα-FERL / b12-FERR was confirmed to have FAPα-specific binding affinity for human and cynomolgus monkey FAPα (Table 6), which was comparable to that of BisG1-FAPα-FERL / DR4-FERR. The control antibody BisG1-b12-FERL / DR4-FERR showed no binding (data not shown).

[0298] [Table 9]

[0299] [Example 6] Binding competition between anti-FAPα antibodies to recombinant human FAPα in a BLI-based classical sandwich cross-block assay Antibody cross-block analysis (epitope binning) was performed using biolayer interferometry (BLI) on an Octet HTX instrument (ForteBio) to determine binding competition for recombinant human FAPα between IgG1-FAPα-F405L and the benchmark FAPα-specific antibodies IgG1-FAP-ESC11-F405L and IgG1-FAP5. IgG1-b12 was included as a negative control.

[0300] Sandwich cross-block experiments were performed at 30°C with shaking at 1,000 RPM. An amine-reactive biosensor (AR2G) (ForteBio, Catalog No. 18-5092) was activated for 300 seconds with a solution of 20 mM EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) (ForteBio, Catalog No. 18-1033) and 10 mM s-NHS (N-hydroxysulfosuccinimide sodium salt) (ForteBio, Catalog No. 18-1067). The activated AR2G sensor was loaded with 10 μg / mL of the first antibody in 10 mM sodium acetate pH 6.0 (ForteBio, Catalog No. 18-1070) for 600 seconds and quenched with 1 M ethanolamine pH 8.5 (ForteBio, Catalog No. 18-1071) for 300 seconds. After a baseline measurement in Sample Diluent (30 seconds; ForteBio, catalog number #18-1048), the AR2G biosensor containing the immobilized antibody was loaded with His-tagged recombinant human FAPα (R&D Systems, catalog number 3715-SE, 100 nM in Sample Diluent) for 200 seconds. The theoretical molecular weight of the His-tagged recombinant human FAPα protein based on the amino acid sequence (86 kDa) was used for calculations. The association (200 seconds) of the second antibody (5 μg / mL in Sample Diluent) was determined. The sensor was regenerated by exposure to 10 mM glycine pH 2.5 (Riedl-deHaen, catalog number #15527) for 5 seconds, followed by neutralization in Sample Diluent for 5 seconds. Both steps were repeated twice. The sensor containing the immobilized first antibody was then used again, starting from the baseline step.

[0301] Data were acquired using Data Acquisition Software v9 (ForteBio) and analyzed with Data Analysis HT Software v9 (ForteBio). The Y-axis was aligned to the onset of the association phase, and Savitzky-Golay filtering was applied. The association response of the second antibody was plotted in matrix format. For each immobilized antibody, responses were corrected by subtracting the average response of two reference sensors incubated with Sample Diluent instead of the second antibody to correct for dissociation of His-tagged recombinant human FAPα protein from the immobilized first antibody. In general, a cutoff of 0.21 nm, based on the typical response to self-blocking, was used to distinguish between blocking antibody pairs (<0.21 nm) and non-blocking antibody pairs (>0.21 nm).

[0302] result Sandwich cross-block experiments using BLI showed that IgG1-FAPα-F405L did not bind to FAPα simultaneously with the benchmark antibody IgG1-FAP-ESC11-F405L (responses below the cutoff, Table 7), indicating that these antibodies block each other's binding in this particular assay setting. The IgG1-FAP5 antibody was able to bind to FAPα simultaneously with either the IgG1-FAPα-F405L or IgG1-FAP-ESC11-F405L antibody (responses above the 0.21 nm cutoff), suggesting that the benchmark IgG1-FAP5 antibody targets a different epitope on FAPα than IgG1-FAPα-F405L. The negative control antibody IgG1-b12 showed no binding.

[0303] [Table 10]

[0304] [Example 7] Species cross-reactivity to DR4 or mouse / rat DR. The binding of BisG1-FAPα-FERL / DR4-FERR, BisG1-DR4-FEAL / b12-FEAR, and IgG1-DR4-FERR to DR4 from various species commonly used in nonclinical toxicology studies was assessed by flow cytometry using ExpiCHO-S cells transiently expressing DR4 from various animal species. In contrast to other species, rodents express only one DR, which has the highest homology to human DR5. Therefore, ExpiCHO-S cells transiently expressing rat and mouse DR proteins were used instead. All DR constructs lack an intracellular DD to avoid apoptosis of transfected cells upon DR(4) activation. IgG1-b12 was used as a negative control.

[0305] ExpiCHO-S cells (Life Technologies, catalog no. EXX8120-3605-036) were transfected with ExpiFectamine™ CHO transfection reagent (Thermo Fisher Scientific, catalog no. A29131), OptiPro™ serum-free medium (Thermo Fisher Scientific, catalog no. 12309019), and ExpiFectamineCHO™ enhancer (Thermo Fisher Scientific, catalog no. A29131) according to the manufacturer's instructions to transfect human (UniProt ID: O00220, lacking aa 365-448 [DD deletion]; SEQ ID NO: 40), cynomolgus monkey (Macaca fascicularis; Uniprot ID: 15309893.2, lacking aa 370-457 [DD deletion]; SEQ ID NO: 41), rabbit (Oryctolagus cuniculus; Uniprot ID: DR4 proteins with a C-terminal HA tag attached to the snorkel domain from dog (Canis familiari; UniProt ID:38280584.1, lacking aa 350-433 [DD deletion]; SEQ ID NO:44), pig (Sus scrofa; UniProt ID:5670488.1, lacking aa 336-423 [DD deletion]; SEQ ID NO:45), or rat (Rattus norvegicus; UniProt ID:B8YBG7, lacking aa 254-323 [DD deletion]; SEQ ID NO:43) and mouse (Mus musculus; UniProt ID:Q9QZM4, lacking aa The cells were transiently transfected with the mammalian expression vector pSB encoding a DR protein lacking residues 273-356 (DD deletion; SEQ ID NO: 42) with a C-terminal HA tag linked to the snorkel domain.

[0306] Binding assays were performed as described in Example 4, with the following modification: 50,000 cells / well were seeded. Binding curves were analyzed using nonlinear regression analysis (four-parameter dose-response curve fitting) in GraphPad Prism.

[0307] result The BisG1-FAPα-FERL / DR4-FERR and BisG1-DR4-FEAL / b12-FEAR antibodies, which can only bind monovalently, demonstrated binding to ExpiCHO-S cells transfected with human DR4 and cynomolgus monkey DR4 (Figures 4A-B). The apparent affinity of BisG1-FAPα-FERL / DR4-FERR for human DR4 was quite high, with a mean EC 50 The mean EC ± SEM was 0.50 ± 0.26 μg / mL for BisG1-FAPα-FERL / DR4-FERR (Table 8), but the mean EC for cynomolgus DR4 was 0.50 ± 0.26 μg / mL because the plateau was not reached. 50 The monoclonal antibody (bivalent binding, IgG1-DR4-FERR) also inhibited the cynomolgus DR4 (mean EC 50 ±SEM 0.14 ± 0.09 μg / mL) compared to human DR4 (mean EC 50 BisG1-FAPα-FERL / DR4-FERR and BisG1-DR4-FEAL / b12-FEAR showed similar dose-dependent binding and EC 50 confirmed that the observed binding was via the DR4-specific arm and not via the FAPα-specific arm.

[0308] No binding to DR4 from rabbit, dog, or pig, or to mouse / rat DRs was observed for BisG1-FAPα-FERL / DR4-FERR or IgG1-DR4-FERR (FIGS. 4C-4G).

[0309] IgG1-b12, used as a negative control, showed no binding to DR4 in any of the species tested (Figures 4A-G).

[0310] In conclusion, the DR4-specific antibody clones used to generate the bispecific antibodies BisG1-FAPα-FERL / DR4-FERR (and BisG1-DR4-FEAL / b12-FEAR) showed cross-reactivity to cynomolgus monkey DR4. However, the apparent affinity for cynomolgus monkey DR4 was lower than that for human DR4. No binding to DR4 from other species (or mouse / rat DR4) was observed (Figure 4C-G).

[0311] [Table 11]

[0312] [Example 8] Binding affinity of DR4 clones to recombinant human and cynomolgus DR4. The binding affinities of BisG1-FAPα-FERL / DR4-FERR, BisG1-b12-FERL / DR4-FERR, and control BisG1-FAPα-FERL / b12-FERR to recombinant human and cynomolgus DR4 proteins were determined using label-free biolayer interferometry on an Octet HTX instrument (Sartorius).

[0313] The protocol detailed in Example 5 was used with the following differences: An anti-Penta-HIS biosensor (Sartorius, Cat. No. 18-5120) was used. The sensor was preconditioned by exposure to 10 mM glycine (Sigma-Aldrich, Cat. No. 15527) buffer pH 1.5 for 5 seconds, followed by neutralization in Sample Diluent (Sartorius, Cat. No. 18-1104) for 5 seconds. Both steps were repeated twice. The sensor was loaded with 50 nM human DR4HsECD-FcHisCtag (SEQ ID NO: 66) or cynomolgus DR4MfECD-FcHisCtag (SEQ ID NO: 67) for 600 seconds. The dissociation time window of interest was set at 100 seconds for cynomolgus antigens and 1,000 seconds for human antigens.

[0314] result BisG1-FAPα-FERL / DR4-FERR showed significantly higher binding affinity to human compared to cynomolgus DR4 (Table 9). A similar nanomolar K was observed with the bispecific antibody with a non-binding control arm, BisG1-b12-FERL / DR4-FERR. D DR4-specific binding was confirmed with values ​​(Table 9). The negative control antibody BisG1-FAPα-FERL / b12-FERR showed no binding (data not shown).

[0315] [Table 12]

[0316] [Example 9] Quantification of DR4, DR5 and FAPα surface expression and assessment of fibroblast cell death. a. Surface expression patterns of FAPα and DR4 on fibroblasts confirm the trans binding of BisG1-FAPα-FERL / DR4-FERR. The proposed mechanism of action of BisG1-FAPα-FERL / DR4-FERR is trans-binding of FAPα expressed on CAFs in the TME and DR4 expressed on tumor cells, resulting in DR4 transactivation-mediated tumor cell death. If cis-binding of BisG1-FAPα-FERL / DR4-FERR to fibroblasts occurs, this could potentially reduce antitumor activity by directly targeting fibroblasts instead of tumor cells. Example 2 demonstrated efficient FAPα binding to lung fibroblasts and CAFs by BisG1-FAPα-FERL / DR4-FERR and IgG1-FAPα-FERL.

[0317] The expression of human FAPα, DR4, and DR5 was assessed in human lung fibroblasts and CAFs by semiquantitative analysis using indirect immunofluorescence staining (Qifi assay). The following primary antibodies were used: mouse anti-human DR4 (Biolegend, catalog no. 307202), mouse anti-human DR5 (Diaclone, catalog no. 854.860.000 for human lung fibroblasts, and Invitrogen, catalog no. 14-9908-82 for CAFs), and mouse anti-human FAPα (USBiological, catalog no. F4208-57E). Human lung fibroblasts were cultured in MEM medium (Lonza, catalog number M5650) supplemented with 10% fetal bovine serum (FBS, ATCC, catalog number 30-2020), 1% L-glutamine (Lonza, catalog number BE17-605E), and 1% penicillin / streptomycin (Pen / Strep, Lonza, catalog number DE17-603E). CAFs were cultured in DMEM high glucose medium (Sigma-Aldrich, catalog number D6429) supplemented with 50 U / mL penicillin / streptomycin, 2 mM GlutaMAX, and 10% FBS (Bodinco BV, catalog number 5067V20002).

[0318] Cell viability was confirmed by AO / PI (Nexcelom, Cat. No. CS2-0106). Human lung fibroblasts and CAFs (50,000 cells / well) were seeded into 96-well round-bottom plates (Greiner Bio-one, Cat. No. 650101). Antibody dilutions were prepared using FACS buffer consisting of PBS (Lonza, Cat. No. BE17-517Q) supplemented with 1% BSA (Roche, Cat. No. 10735086001) and 0.02% sodium azide (Bio-World, Cat. No. 41920044-3). For experiments using CAFs, the following steps were performed. Plates were centrifuged, the supernatant removed, and cells were resuspended in 50 μL of human Fc Block (BD, catalog number 564220, diluted 1:100 in FACS buffer) and 50 μL of viability stain TO-PRO-3 Iodide (Thermo Fisher, catalog number T3605, diluted 1:25,000 in FACS buffer) and incubated for 15 minutes at 4°C. Plates were washed once with FACS buffer. Plates containing either human lung fibroblasts or CAFs were centrifuged, the supernatant removed, and cells were resuspended in 50 μL of primary antibody (final concentration of 10 μg / mL in FACS buffer) and incubated for 30 minutes at 4°C. Cells were washed three times with FACS buffer and resuspended in 50 μL of FITC-labeled polyclonal antibody goat anti-mouse IgG1 (Dako, catalog number F047902-2, diluted 1:50 for experiments using CAFs and 1:100 for experiments using human lung fibroblasts) in FACS buffer. In parallel, 15 μL of human-constituted Qifi beads (Biocytex, catalog number CP010) and 15 μL of calibrated Qifi beads (Dako, catalog number K0078) were added to empty wells. After 30 min of incubation at 4 °C, cells were washed twice with FACS buffer and resuspended in FACS buffer. All samples were analyzed on an iQue flow cytometer (Sartorius) or a FACSCelesta flow cytometer (BD biosciences), and data were processed using FlowJo_v10.8.1 (FlowJo LLC). Data were analyzed using GraphPad Prism.

[0319] In the Qifi assay, the primary antibody was used at saturating concentrations, and the number of bound primary antibody molecules corresponds to the number of antigen sites present on the cell surface. A FITC-conjugated secondary antibody was also used at saturating concentrations to correlate fluorescence intensity with the number of bound primary antibody molecules on cells and beads. The recorded fluorescence values ​​from the calibration beads, along with a clearly defined number of IgG monoclonal antibodies per bead, were used to generate a standard curve using GraphPad Prism software. The software then used the standard curve equation to calculate the specific antibody binding capacity (sABC, corresponding to the average number of accessible antigen or molecule sites per cell) of the antibody-stained cells.

[0320] result Human lung fibroblasts expressed high levels of human FAPα and low levels of DR5 (25,387 ± 2,564 and 5,841 ± 479 molecules per cell, respectively; mean ± SEM of two independent experiments) (Figure 5A). DR4 expression was below the lower limit of quantification (LLOQ, dashed line, Figure 5A). Similar results were observed for CAFs: high FAPα expression (109,759 ± 25,397 molecules / cell, mean ± SEM of three independent experiments), low DR5 expression (11,610 ± 502 molecules / cell, mean ± SEM of two independent experiments), and DR4 expression below the LLOQ (Figure 5B). In conclusion, the binding data from Example 2 and these results suggest that BisG1-FAPα-FERL / DR4-FERR targets only CAFs bearing the FAPα arm, but that DR4 activation on fibroblasts as a result of simultaneous binding to DR4 and FAPα is unlikely.

[0321] b. BisG1-FAPα-FEAL / DR4-FEAR does not induce cell death in fibroblasts. Next, we evaluated the ability of BisG1-FAPα-FEAL / DR4-FEAR, BisG1-FAPα-FEAL / b12-FEAR, and BisG1-b12-FEAL / DR4-FEAR to induce death of human lung fibroblasts and CAFs. IgG1-b12-FEAR (negative control), IgG1-FAPα-FEAL, and RG7386 were also tested.

[0322] Human lung fibroblasts were detached using trypsin / EDTA (Lonza, catalog no. 17-161E) and viability was confirmed using AO / PI (Nexcelom, catalog no. CS2-0106-25ml). 100 µL containing 5,000 cells / well was added to a 96-well flat-bottom plate (Greiner Bio-One, catalog no. 655180). After 24 h incubation at 37 °C and 5% CO2, 50 µL / well of antibody dilutions (10–0.0000256 µg / mL, 5-fold dilutions in FACS buffer) were added. After 72 h incubation at 37 °C and 5% CO2, 15 µL / well of Cell-Titer Glo (Promega, catalog no. G7571) was added. After further incubation of the plate at 37°C and 5% CO2 for 1.5 hours, 100 μL of supernatant was transferred to a 96-well white OptiPlate (Perkin Elmer, catalog no. 6005299). Luminescence (viability readout) was measured using an Envision instrument. Data were analyzed and visualized using GraphPad Prism. The percentage of viable cells normalized to the no-antibody condition was plotted against antibody concentration.

[0323] Follow-up experiments using human lung fibroblasts were performed with the following modifications: Trypan blue (Sigma, Catalog No. T8154-100ml) was used to confirm cell viability. 96-well flat-bottom plates were from Falcon® (Cat. No. 353072). After 3 hours of incubation, serial antibody dilutions (1-0.000002 μg / mL, 5-fold serial dilutions using M0130 medium) were added. Reagents were diluted using M0130 medium, consisting of RPMI (Gibco, Catalog No. A10491-01) supplemented with 10% heat-inactivated donor bovine serum (Gibco, Catalog No. 20371-030). As a positive control for killing, 3 μM staurosporine / well was added, and antibody IgG1-b12 was used as a negative control for killing. Next, 50 μL / well of 100 nM Cytotox Green (Essenbio, catalog number 4633, diluted in M0130) was added, and the plates were incubated in Incucyte for 72 hours (37°C, 5% CO2) with images taken every 3 hours. Data were generated and processed using Incucyte software 2021B. Data were analyzed and visualized using GraphPad Prism. Data were fitted with a nonlinear 4-parameter logistic curve. For each condition at each time point, the Incucyte software calculated the number of Cytotox Green-positive dead cells, which were plotted against time.

[0324] For experiments with CAFs, CAFs were detached using trypsin (Lonza, catalog no. BE02-007E) and counted using trypan blue (Fluka, catalog no. 93590). 10,000 CAFs / well were seeded onto a collagen (Ibidi, catalog no. 50204) monolayer (7.5 μg / mL collagen in 17.5 mM acetic acid, from VWR, catalog no. 30010.292) in a 96-well plate and incubated at 37°C and 5% CO for 24 h. After this, 12 μL / well of BisG1-FAPα-FEAL / DR4-FEAR antibody dilutions (20–0.05 μg / mL, 4-fold dilutions in FACS buffer) were added. After 72 hours at 37°C, 5% CO2, cell viability was assessed by adding 80 μL / well of prewarmed cell TiterGlo3D (Promega, catalog number G9681) to each well. Luminescence was read after 30 minutes (of which the first 15 minutes were gently agitated) using a Spectramax plate reader (Molecular Devices). Data were processed and visualized using GraphPad Prism. Data were fitted with a nonlinear 4-parameter logistic curve. Graphs show the % viable tumor cells ± SEM of technical replicates, normalized to the no-antibody condition and plotted against antibody concentration.

[0325] result In two separate experiments using human lung fibroblasts, BisG1-FAPα-FEAL / DR4-FEAR, IgG1-FAPα-FEAL, BisG1-FAPα-FEAL / b12-FEAR, or BisG1-b12-FEAL / DR4-FEAR did not alter lung fibroblast survival when compared with the negative control IgG1-b12-FEAR (Figure 5C-D). However, treatment with high concentrations of RG7386 demonstrated a decrease in fibroblast survival (Figure 5C). CAF survival was also unaffected by treatment with BisG1-FAPα-FEAL / DR4-FEAR (Figure 5E). These results indicate that the BisG1-FAPα-FEAL / DR4-FEAR antibody does not induce fibroblast cell death.

[0326] [Example 10] Transactivation ability of BisG1-FAPα-FEAL / DR4-FEAR a. Comparison between FAPα-binding clones in a bispecific format to induce DR4 transactivation-mediated cell death. The ability of BisG1-FAPα-FEAL / DR4-FEAR and BisG1-FAP5-FEAL / DR4-FEAR to induce DR4 transactivation-mediated cell death of the DR4-expressing human cancer cell line MDA-MB-231 in the presence of human lung fibroblasts was evaluated. BisG1-FAPα-FEAL / b12-FEAR, BisG1-FAP5-FEAL / b12-FEAR, and BisG1-b12-FEAL / DR4-FEAR were used as negative controls.

[0327] In vitro viability assays were performed using the human cancer cell line MDA-MB-231 (breast cancer, ATCC, catalog number HTB26) cultured in the presence of human lung fibroblasts. MDA-MB-231 cells were cultured in M0089 medium (consisting of DMEM with high glucose and HEPES [N'-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid] (Lonza, catalog number BE12-709F) supplemented with 10% donor bovine serum with iron (Life Technologies, catalog number 20371), 1% L-glutamine (Lonza, catalog number BE17-605E), 1% 100 mM sodium pyruvate (Lonza, catalog number BE13-115E), and 1% 100x non-essential amino acid solution (Life Technologies, catalog number 11140) containing 0.3 μM CytoLight Rapid Red (Essen). The cells were incubated with 1 μM stock (Gibco, Cat. No. 4706) at 37°C for 20 min and washed three times with culture medium M0130 (RPMI (Gibco, Cat. No. A10491-01) supplemented with 10% heat-inactivated donor bovine serum (Gibco, Cat. No. 20371-030)). The viability of MDA-MB-231 cells was confirmed with trypan blue (Sigma-Aldrich, Cat. No. T8154-100ml). Cells (5,000 CytoLight Rapid Red-labeled cells / well and 2,500 human lung fibroblasts / well) were seeded in 96-well flat-bottom plates (Falcon, catalog no. 353072) in M0130 medium and incubated at 37 °C and 5% CO for 24 h. Then, serial antibody dilutions (1000 to 0.00051 ng / mL final concentrations, 5-fold serial dilutions in M0130 medium) were added. Negative control antibodies (BisG1-FAPα-FEAL / b12-FEAR, BisG1-FAP5-FEAL / b12-FEAR, and BisG1-b12-FEAL / DR4-FEAR) were added at a fixed concentration of 1000 ng / mL. Staurosporine (Sigma, catalog no. S6942, diluted in M0130 medium) was used as a positive killing control.Next, 50 μL / well of 100 nM Cytotox Green (Essen Bio, Cat. No. 4633, diluted in M0130 medium) was added, and the co-culture plates were then incubated in an Incucyte® (Essenbio) at 37°C and 5% CO2 for 72 hours, with imaging every 4 hours. Data were generated and processed using Incucyte software 2021B. Data were analyzed and visualized using GraphPad Prism. Data were fitted with a nonlinear 4-parameter logistic curve. For each condition, the Incucyte software calculated a signal (overlapped area, μm) corresponding to the number of Cytotox Green-positive dead cells for each time point. 2 / image) and the signal corresponding to the number of total cells (red target area, μm 2 The mean (p < 0.05) / image was calculated. Viable tumor cells were calculated by subtracting the signal of dead cells from the signal of total cells. The area under the curve (AUC) was then calculated using GraphPad Prism and plotted against antibody concentration.

[0328] result In cocultures of MDA-MB-231 cells and human lung fibroblasts, BisG1-FAPα-FEAL / DR4-FEAR and BisG1-FAP5-FEAL / DR4-FEAR induced dose-dependent cell death (Figure 6A). BisG1-FAPα-FEAL / DR4-FEAR induced maximal DR4-dependent MDA-MB-231 cell death at lower concentrations than BisG1-FAP5-FEAL / DR4-FEAR (Table 10). As expected, the control bispecific antibodies BisG1-FAPα-FEAL / b12-FEAR, BisG1-FAP5-FEAL / b12-FEAR, and BisG1-b12-FEAL / DR4-FEAR did not induce cell death.

[0329] [Table 13]

[0330] b. BisG1-FAPα-FERL / DR4-FERR induces DR4 transactivation-mediated cell death only in the presence of FAPα-expressing cells. The ability of BisG1-FAPα-FERL / DR4-FERR to induce DR4 transactivation-mediated cell death of DR4-expressing human cancer cells was assessed in the presence and absence of FAPα-expressing cells. The BisG1-b12-FERR / b12-FERL antibody was used as a negative control. RG7386 was included in experiments using cancer cells alone, and the control antibodies BisG1-b12-FERL / DR4-FERR and BisG1-FAPα-FERL / b12-FERR were included in coculture experiments.

[0331] For coculture experiments, cancer cells were cultured in the presence of the cell line NIH / 3T3 (mouse fibroblasts, ATCC, catalog number CRL-1658) transfected to express the FAPα mature polypeptide (SEQ ID NO: 33) as follows: On the day of transfection, NIH / 3T3 cells were harvested using 0.5% trypsin / EDTA (Gibco, catalog number 25300-062) and viability was measured using a Vicell-BLU instrument (Beckman Coulter). A total of 1 million cells were transferred to a 15 mL tube (Greiner Bio-one, catalog no. 188271), washed with Hank's Balanced Salt Solution (HBSS, Gibco, catalog no. 14175-053), resuspended in 100 μL of 4D-Nucleofector solution (500 μL of Supplement 1 mixed with 2.25 mL of SG Cell Line Solution; both reagents are from the SG Cell Line Solution box, Lonza, catalog no. PBC3-02250), and transferred to a cuvette (Lonza, catalog no. PCK-2005) containing 4.7 μL of pGENPGK-FAP-puro plasmid DNA. The pGENPGK-FAP-puro expression vector (approximately 7,000 bp in size) contained the following key elements: full-length human FAP, expression of which was driven by the phosphoglycerate kinase (PGK) promoter; a puromycin selection marker, expression of which was driven by the simian virus 40 (SV40) promoter; a Kozak sequence upstream of the PGK promoter, expression of which was driven by the beta-lactamase (BLA) promoter, and an ampicillin selection marker. Electroporation was performed using a 4D-Nucleofector apparatus (X unit, Lonza). Next, 400 μL of M0057-05 medium, consisting of DMEM high glucose and HEPES (Lonza, catalog number BE12-709F) supplemented with 10% heat-inactivated donor bovine serum with iron (DBSI, (Life Technologies, catalog number 20371)) and 1% 200 mM L-glutamine (Life Technologies, catalog number 25030-081), was added to the cuvette.After 10 minutes of incubation at 37°C and 5% CO2, the cuvette contents were transferred using a Pasteur pipette (from a Lonza kit, catalog number V4XC-3024) to a 24-well plate (Cellstar, catalog number 662 160) already containing 500 μL / well of M0057-05 medium. The plate was kept in an incubator (37°C, 5% CO2) to allow the cells to recover and grow. After 72 hours, the medium was replaced with 1 mL / well of medium consisting of 3.5 μL of puromycin (stock solution 10 mg / mL, Sigma, catalog number P9620) added to 20 mL of a 1:1 mixture of fresh M0057-05 medium and M0057-05 medium harvested from a flask containing 100% confluent, untransfected NIH / 3T3 cells. The medium change step was repeated every 4 days until enough cells were available for evaluation of transfection efficiency (FAPα expression) by flow cytometry.

[0332] To measure FAPα expression, NIH / 3T3-FAPα cells were seeded at 10,000 / well (round-bottom 96-well plates), centrifuged, and resuspended in 20 μL / well of IgG1-FAPα-FERL primary antibody (5 mg / mL stock concentration, diluted 1:4 in FACS buffer). After 20 min of incubation at 4°C in the dark, cells were washed twice with FACS buffer and resuspended in 20 μL of secondary FITC-labeled polyclonal antibody goat anti-mouse IgG1 (diluted 1:4 in FACS buffer), followed by an additional 20 min incubation step (4°C in the dark). Cells were washed twice with FACS buffer, resuspended in FACS buffer, and read using CellStream (Luminex). FAPα expression measured by gMFI was comparable to that measured for a positive control (HEK293F cells transiently expressing FAPα) (data not shown).

[0333] In vitro survival assays were performed using two human cancer cell lines: DLD-1 (colorectal adenocarcinoma, ATCC, catalog no. CCL221) cultured in M0130 medium and MDA-MB-231 (cultured in M0089 medium). Cells were harvested using trypsin (Gibco, catalog no. 25300-054), and cell viability was measured using AO / PI (Nexcelom, catalog no. CS2-0106). 50 μL of cancer cells (6,600 / well) were seeded into a 96-well plate (Perkin Elmer, catalog no. 6005680) with or without 50 μL of NIH / 3T3-FAPα cells (3,300 / well). After incubating the cells for 4 hours (37°C, 5% CO2) to allow them to adhere to the plate, 50 μL / well of an antibody concentration series (final concentrations of 14.4–0.000007 μg / mL, diluted 8-fold in M0130 medium) was added. 50 μL / well of phenylarsine oxide (PAO, Sigma-Aldrich, catalog number P3075, stock concentration 50 mg / mL diluted 1:1,000 in FACS buffer) was used as a positive control for killing. The plate was incubated for 72 hours (37°C, 5% CO2) before viability was read. For viability readout, 20 μL / well of cell TiterGlo (Promega, catalog number G755A) was added, and the plate was incubated for 1.5 hours (37°C, 5% CO2) before luminescence was read using EnVision. The percentage of viable cells was calculated using the following formula: ([Signal Sample - Signal PAO Control] - [Signal Fibroblasts Only - Signal PAO Control]) / ([Signal Cancer Cells Only - Signal PAO Control] - [Signal Fibroblasts - Signal PAO Control]). Data were analyzed and visualized using GraphPad Prism. Data were fitted with a nonlinear 4-parameter logistic curve. The percentage of viable cells (in duplicate) is plotted against antibody concentration.

[0334] result In monocultures of MDA-MB-231 and DLD-1 cancer cells, survival after treatment with BisG1-FAPα-FERL / DR4-FERR was comparable to that of the control BisG1-β12-FERL / β12-FERR. Treatment with the highest concentration of RG7386, on the other hand, reduced cancer cell survival (Figures 6B-C; Table 1). Both MDA-MB-231 and DLD-1 cancer cells express DR5 (data not shown).

[0335] [Table 14]

[0336] In cocultures of cancer cells with NIH / 3T3-FAPα effector cells, BisG1-FAPα-FERL / DR4-FERR induced dose-dependent cell death (Figures 6D-6E; Table 12 for the top four concentrations). As expected, BisG1-b12-FERL / DR4-FERR, BisG1-FAPα-FERL / b12-FERR, and BisG1-b12-FERL / b12-FERR (all containing at least one nonbinding control Fab arm) did not induce substantial tumor cell death. Thus, DR4 transactivation-mediated cancer cell death depends on targeting of DR4 and FAPα by the bispecific antibody.

[0337] [Table 15]

[0338] c. Caspase-8 activation To assess signaling downstream of DR4 transactivation, caspase-8 activation was assessed in cocultures of tumor cell lines with NIH / 3T3-FAPα cells in the presence or absence of BisG1-FAPα-FERL / DR4-FERR, RG7386, positive control recombinant human TRAIL (Biolegend, catalog no. 752906), and negative control antibody BisG1-b12-FERL / DR4-FERR.

[0339] DR4-expressing tumor cells (MDA-MB-231, A549, DLD-1, and SNU-1076 [head and neck cancer; Creative Bioarray catalog number CSC-C9620L]) were harvested as described in Example 3. NIH-3T3-FAPα cells were harvested as described in section b of Example 10. Tumor cells and fibroblasts were seeded at a 2:1 ratio (13,300 tumor cells and 6,700 fibroblasts / well) into White Opaque 96-well microplates (PerkinElmer, catalog number 6005680) and incubated overnight at 37°C and 5% CO2 to allow cells to adhere to the plate. Next, an antibody concentration series (0.0003 to 100 nM in 5-fold increments) was added. Samples were mixed on a plate shaker (300 RPM) for 2 minutes, and the plate was incubated at 37°C and 5% CO2 for 5 hours.

[0340] Activation of the extrinsic apoptotic pathway was determined by measuring caspase-8 activation using a homogeneous Caspase-Glo® 8 luminescence assay (Promega, catalog number G8202). Plates were first left at room temperature for 30 minutes. Then, 100 μL / well of Caspase-Glo 8 reagent from the kit was added, mixed on a plate shaker (300 RPM) for 2 minutes, and incubated at room temperature for 1 hour in the dark. Luminescence was measured using an EnVision Multiplate Reader. Luminescence data were processed with GraphPad Prism software to generate dose-response curves using nonlinear regression analysis (sigmoidal dose-response with variable slope).

[0341] result Dose-dependent caspase-8 activation for BisG1-FAPα-FERL / DR4-FERR was observed in cocultures of DLD-1, MDA-MB-231, A549, and SNU-1076 tumor cells with NIH / 3T3-FAPα cells (Figures 7A, 7C, 7E, and 7G), but not in monocultures (Figures 7B, 7D, 7F, and 7H). These data suggest that BisG1-FAPα-FERL / DR4-FERR-induced cytotoxicity is mediated by caspase-8 activation. The maximum caspase-8 activation levels observed were comparable for BisG1-FAPα-FERL / DR4-FERR and TRAIL, which was included as a positive control. Furthermore, dose-dependent caspase-8 activation was observed for RG7386 in all tumor cell lines (Figure 7).

[0342] The lack of BisG1-FAPα-FERL / DR4-FERR-induced caspase-8 activation in tumor cell monocultures or cocultures in the presence of BisG1-b12-FERL / DR4-FERR indicates that caspase-8 activation by BisG1-FAPα-FERL / DR4-FERR is conditional and dependent on both DR4 and FAPα binding. In contrast, the unconditional agonist TRAIL induced caspase-8 activation even in tumor cell monocultures, independent of the presence of FAPα. Limited caspase-8 activation was further observed in tumor cell monocultures containing RG7386 (potentially due to its ability to bivalently bind to DR5), but not in BisG1-FAPα-FERL / DR4-FERR (Figures 7A-H).

[0343] In summary, BisG1-FAPα-FERL / DR4-FERR exhibits efficient caspase activation in all tumor cell lines tested that is dependent on the presence of FAPα-expressing fibroblasts, consistent with the conclusions of transactivation-mediated cell death presented in sections a and b of this example.

[0344] [Example 11] Targeted cell death of PDO via DR4 transactivation in the presence of CAFs The ability of BisG1-FAPα-FEAL / DR4-FEAR to induce DR4 transactivation-mediated cell death through trans-binding of CRC PDO was investigated in the presence and absence of CAFs. IgG1-FAPα-FEAL was used as a negative control.

[0345] In vitro survival assays were performed using PDO from three patients with CRC: Hub096 (primary tumor, ascending colon), p19B (primary tumor, ascending colon), and p18T (primary tumor, sigmoid). PDO were cultured in a basement membrane extract (BME) matrix (Amsbio, catalog number 3533-010-02) supplemented with the following supplements: 10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) buffer (Lonza, catalog number 17737E), 50 U / mL penicillin / streptomycin (Gibco, catalog number 15070-063), 2 mM GlutaMAX (Gibco, catalog number 35050-038), 20% R-spondin conditioned medium (293T-HA-Rspol-F cell line), 100 ng / mL erythrocyte sedimentation medium (293T-HA-Rspol-F cell line). Noggin-conditioned medium (293T-mNoggin-Fc cell line), 1x B27 (Invitrogen, catalog no. 17504-044), 10 mM nicotinamide (Sigma-Aldrich, catalog no. N0636), 10 nM prostaglandin E2 (Tocris, catalog no. 2296-10), 10 nM gastrin (Sigma-Aldrich, catalog no. G9145), 0.5 mM N-acetylcysteine ​​(NAC, Sigma-Aldrich, catalog no. A9165), 500 nM A83-01 (SignalChem, catalog no. A09-900-05), 50 ng / mL human recombinant epidermal growth factor (EGF, Sigma-Aldrich, catalog no. A9165), 10 μM SB202190 (p38 inhibitor, Gentaur, catalog no. A1632), and 10 mM CAFs were cultured at a 2:1 ratio in PDO medium consisting of Advanced DMEM / F12 medium (Gibco, catalog no. 12634-010) containing Y27632 (Rock kinase inhibitor, Abmole Bioscience, catalog no. HY-10583). CAFs were cultured in CAF medium consisting of DMEM high glucose medium (Sigma-Aldrich, catalog no. D6429) supplemented with 50 U / mL penicillin / streptomycin, 2 mM GlutaMAX, and 10% FBS (Bodinco BV, catalog no. 5067V20002).

[0346] CellTiter-Glo viability assay with PDO and CAFs The day before coculture experiments, CAFs were detached using trypsin (Lonza, catalog no. BE02-007E) and counted using trypan blue (Fluka, catalog no. 93590). 10,000 CAFs per well were seeded onto a collagen (Ibidi, catalog no. 50204) monolayer (7.5 μg / mL collagen in 17.5 mM acetic acid, from VWR, catalog no. 30010.292) in a 96-well plate (Thermo Fisher Scientific, catalog no. 165306) and incubated at 37°C and 5% CO2. The next day, PDOs were harvested and dissociated using TripLE™ Express (Gibco, catalog no. 12604021) at 37°C for 5 minutes. Cells were washed with PBS (Corning, catalog no. 21-0310CVR), counted using trypan blue, and resuspended in 5% Matrigel (Corning, catalog no. 354234) diluted in co-culture medium consisting of Advanced DMEM / F12 supplemented with HEPES buffer, penicillin / streptomycin, GlutaMAX, B27, EGF, Y27632, A83-01, and NAC (as above), 10 ng / mL human insulin (Sigma-Aldrich, catalog no. 19278), 10 ng / mL human fibroblast growth factor (FGF)-basic (Prepotech, catalog no. 100-18B), and 25 ng / mL platelet-derived growth factor receptor (PDGFR) α / β (Sigma-Aldrich, catalog no. p3326). 10,000 PDO was added to empty wells or to the CAF monolayer, and the plates were incubated at 37°C and 5% CO2 for 4 hours, followed by the addition of 12 μL / well of an antibody concentration series (10–0.001 μg / mL, 5-fold serial dilutions in co-culture medium). All conditions were tested in technical replicates. After 72 hours at 37°C and 5% CO2, cell viability was assessed by adding 80 μL / well of prewarmed TiterGlo3D (Promega, catalog number G9681) to each well. After 30 minutes (of which the first 15 minutes were gently agitated), luminescence was read using a Spectramax plate reader (Molecular Devices). Data were processed and visualized using GraphPad Prism.Data were fitted with a nonlinear 4-parameter logistic curve. Graphs show % viable tumor cells ± SEM in duplicates normalized to the PDO-only condition (no CAFs or antibody) and plotted against antibody concentration.

[0347] Annexin V viability assay The day before the start of the coculture experiment, CAFs were harvested as described for the CellTiter-Glo assay and incubated with the cytoplasmic membrane dye CellBrite Orange (Biotium, Cat. No. 30022) for 1.5–2 h at 37 °C and 5% CO. Cells were then washed three times with CAF medium and plated at 4 × 10 cells per well onto collagen-coated 6-well plates (Costar®, Cat. No. 3506). 5 PDO Hub096 cells were plated at 5 × 10 cells / well (see CellTiter-Glo assay instructions for coating procedure). The next day, PDO Hub096 cells were harvested as described for the CellTiter-Glo assay, and then the single-cell suspension was incubated with the cytoplasmic membrane dye CellBrite Blue (Biotium, Cat. No. 30024) for 1.5–2 h at 37°C and 5% CO2. Cells were then washed, resuspended in PDO medium supplemented with 5% Matrigel, and plated at 5 × 10 cells on top of adherent CellBrite Orange-labeled CAFs. 5 Cells / well were seeded and incubated with antibody samples (0.1 μg / mL or 0.02 μg / mL) overnight at 37° C. and 5% CO 2 .

[0348] After overnight incubation, cells were harvested using trypsin, washed with cold PBS, and gently resuspended in 200 μL of Annexin-V-FITC master mix consisting of 125 μL of Annexin-V-FITC (BD Pharmingen™, Cat. No. 556419) and 2.5 mL of 1× binding buffer (BD Pharmingen, Cat. No. 556454). Cells were incubated with the Annexin-V mix for 30 minutes at room temperature in the dark. Next, 400 μL of 1× binding buffer was added to each tube and transferred to a 5 mL FACS tube (Falcon®, Cat. No. 352008). Annexin-V positivity was measured by flow cytometry on a FACSCelesta Cell Analyzer (BD Biosciences) and FITC detection in the CellBrite Orange-positive CAF and CellBrite Blue-positive PDO cell populations was determined using the FACSCelesta Cell Analyzer Software. + Cells were analyzed by gating.

[0349] result BisG1-FAPα-FEAL / DR4-FEAR induced dose-dependent cell death in all three PDOs, although sensitivity to BisG1-FAPα-FEAL / DR4-FEAR varied among the PDOs tested. Cell death was observed only in the presence of CAFs. Treatment of PDO monocultures did not result in DR4 transactivation-mediated cell death, confirming that the bispecific antibody requires dual binding to DR4 and FAPα to induce cell death. Treatment with the negative control IgG1-FAPα-FEAL antibody did not result in PDO cell death (Figures 8A-8C; Tables 13-15).

[0350] Coculture of fluorescently labeled PDO line Hub096 with fluorescently labeled CAFs confirmed that BisG1-FAPα-FEAL / DR4-FEAR induced an apoptotic phenotype only in PDO Hub096 (as assessed by Annexin V positivity) without affecting CAF survival (Figure 8D, Table 16).

[0351] [Table 16]

[0352] [Table 17]

[0353] [Table 18]

[0354] [Table 19]

[0355] [Example 12] Evaluation of BisG1-FAPα-FEAL / DR4-FEAR antitumor activity in vivo a. Tumor tissues from patients express various levels of FAPα To demonstrate its mechanism of action, BisG1-FAPα-FEAL / DR4-FEAR requires expression of both FAPα and DR4 in the TME to enable trans-binding-dependent DR4 agonism, leading to tumor cell death. For in vivo proof-of-concept studies, two patient-derived xenograft (PDX) models with known DR4 mRNA expression, CTG-1234 (gastric) and CTG-1150 (pancreatic), available from the vendor, were evaluated for FAPα expression using immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded (FFPE) tissue slides.

[0356] FFPE tissue from a patient with invasive ductal carcinoma (Avaden Biosciences) was used as a positive control. Tissue sections were transferred to Superfrost Plus glass slides (Fisher Scientific; catalog number 10149870) and subjected to IHC staining using a Ventana Discovery-Ultra (Roche) platform, along with FFPE tissue slides from CTG-1234 and CTG-1150 (both from Champions Oncology). The tissue staining was initiated with a 37°C incubation to warm the slides, followed by a baking step (60°C for 12 minutes). Next, three deparaffinization cycles were performed, each consisting of an 8-minute incubation at 70°C, a 1 / 10 dilution with EZ Prep (Roche, catalog number 05279755001), a 4-minute coverslip, and EZ Prep application. After the third cycle, the slides were again washed with EZ Prep and loaded with EZ Prep. The slides were incubated at 37°C and washed, after which Discovery Cell Conditioner 1 (CC1; Roche, catalog number 06414575001) reagent was applied for antigen retrieval. The slides were incubated at 95°C for 40 minutes. The slides were then incubated in CC Medium Coverslip (LCS; Roche, catalog number 05264839001) for 16 minutes. The slides were placed at 37°C and washed three times with reaction buffer (Roche, catalog number 05353955001), after which one drop of inhibitor CM (Roche, catalog number 07017944001) was applied and incubated for 8 minutes. After washing the slides twice with reaction buffer, the primary antibody rabbit anti-FAPα clone EPR20021 (reactive with both human and mouse FAPα: final concentration 5 μg / mL, Abcam, catalog no. ab207178) or rabbit IgG isotype control (final concentration 5 μg / mL, Cell Signaling Technology, catalog no. 3900S) was added.Slides were incubated for 32 minutes, washed twice, and then incubated with OmniMap anti-rabbit HRP-conjugated secondary antibody (Roche, catalog number 05269679001) for 16 minutes. After three washing steps, slides were incubated with ChromoMap 3,3'-diaminobenzidine (DAB) and ChromoMap H2O2 for 8 minutes, followed by ChromoMap Copper for 4 minutes (all included in the ChromoMap DAB kit from Roche, catalog number 05266645001). Slides were washed twice and then incubated with Hematoxylin II (Roche, catalog number 05277965001) for 12 minutes and Bluing reagent (Roche, catalog number 05266769001) for 8 minutes. Slides were washed three times and coverslipped using Epredia™ ClearVue™ mounting medium (Fisher Scientific, Cat. No. 23-425-401).

[0357] Immunostained FFPE tissue slides were scanned at 20x magnification using an AxioScan slide scanner (Zeiss). For scoring of FAPα-positive PDX tumor tissue, image scans were analyzed with HALO software (Indica Labs) to quantify the percentage of FAPα-positive tissue surface area and IHC intensity using the predesigned Area Quantification v2.4.2 image analysis algorithm.

[0358] result FAPα positivity was demonstrated for both PDX tissues (IHC intensity staining was low (1+) to moderate (2+) over most of the surface area; Figure 9A), indicating that both PDX models were considered suitable for in vivo proof-of-concept studies.

[0359] b. Antitumor activity in the gastric PDX CTG-1234 model The ability of BisG1-FAPα-FEAL / DR4-FEAR to induce antitumor activity in a gastric cancer-derived PDX model was evaluated in mice. BisG1-DR4-FEAL / b12-FEAR was used as a negative control.

[0360] Stock mice (Envigo's Athymic Nude-Foxn1nu) were implanted bilaterally with fragments of Champions TumorGraft® CTG-1234, originally derived from human gastric cancer biopsies. Tumors were 1000-1500 mm 3 After reaching 100 μg / mL, they were harvested and tumor fragments were implanted subcutaneously into the left flank of female test mice (Envigo athymic Nude-Foxn1nu, 6-8 weeks old at the start of the experiment).

[0361] Monitor tumor growth twice a week using digital calipers and calculate the tumor size using the formula 0.52 × (length × width) 2 The tumor volume (TV) was calculated using the RT-PCR method. The study was conducted with a mean tumor volume (MTV) of 1500 mm in surviving mice (uncensored) in the control group. 3 It ended when it reached

[0362] TV is about 200mm 3 Once tumor size reached 100 mg / mL, animals were matched by tumor size and assigned to control or treatment groups (n=8 / group), with dosing beginning on day 0. On the treatment day, mice were injected intravenously (IV) with BisG1-FAPα-FEAL / DR4-FEAR (0.5 mg / kg, 2 mg / kg, or 8 mg / kg, given once a week for 3 weeks [QW x 3]) or control BisG1-DR4-FEAL / b12-FEAR (8 mg / kg; QW x 3) (Table 17).

[0363] Mice were monitored daily for clinical signs of disease and weighed twice weekly using a digital scale. Data including individual and mean gram weights and mean percent weight change relative to day 0 (%vD0) were recorded for each group. Animal deaths, if any, were recorded. Groups reporting a mean loss of %vD0 greater than 20 and / or a mortality rate greater than 10% were considered to have exceeded the maximum tolerated dose (MTD) for that treatment with the evaluated regimen. Additional study toxicity endpoints were when mice were moribund or showed a net weight loss of >20% over a consecutive 7-day period, or when mice showed a net weight loss of >30%.

[0364] Tumor growth inhibition (TGI) was determined by calculating the percent TGI (100% × [1 - (final MTV - initial MTV of treatment group) / (final MTV - initial MTV of control group)]). Tumor volumes of treatment groups during and at the end of the study were compared with those of the control group. Mean tumor volumes on day 42 (the final day after all groups were terminated) were used to analyze statistical differences between the various treatment groups (Mann-Whitney method).

[0365] One additional endpoint used to evaluate efficacy was progression-free survival (PFS). Kaplan-Meier curves were analyzed using the log-rank (Mantel-Cox) test to assess statistical significance of differences in PFS time (500 mm). 3 tumor size cutoff was used).

[0366] [Table 20]

[0367] result Rapid tumor outgrowth was observed in tumor-bearing mice treated with the negative control BisG1-DR4-FEAL / b12-FEAR (Figure 9B). BisG1-FAPα-FEAL / DR4-FEAR significantly inhibited tumor growth of gastric cancer PDX tumors in athymic nude mice at all doses tested (Table 18 and Figure 9D, p = 0.0002 for all doses tested). PFS in this model was significantly prolonged by BisG1-FAPα-FEAL / DR4-FEAR treatment, even at the lowest dose of 0.5 mg / kg, demonstrating strong antitumor activity (p < 0.0001; Mantel-Cox; Figure 9C). Finally, body weight measurements over time showed no change compared to the negative control group, indicating that treatment was well tolerated (Figure 9E).

[0368] [Table 21]

[0369] c. Antitumor activity in the pancreatic PDX CTG-1150 model A parallel experiment was performed in which athymic nude mice were implanted with Champions TumorGraft® CTG-1150, originally derived from a human pancreatic cancer biopsy. The same treatment regimens, protocols, and statistical tests as described in section b (and Table 17) were applied, with the following differences: statistical comparison of tumor volumes was performed on day 25 (the last day all groups were completed), and PFS was assessed at 1000 mm. 3 A tumor size cutoff of 100 mg / kg was used.

[0370] result As observed in the gastric PDX model, rapid tumor outgrowth was observed in mice bearing pancreatic PDX treated with BisG1-DR4-FEAL / b12-FEAR antibody (Figure 9H). BisG1-FAPα-FEAL / DR4-FEAR administered at 2 mg / kg significantly inhibited tumor growth in the athymic nude mouse pancreatic PDX model compared with the BisG1-DR4-FEAL / b12-FEAR control (Table 19 and Figure 9F, p = 0.038). BisG1-FAPα-FEAL / DR4-FEAR treatment did not prolong PFS compared with the BisG1-DR4-FEAL / b12-FEAR control (Figure 9G). Finally, body weight measurements over time showed no change, indicating that the treatment was well tolerated (Figure 9I).

[0371] [Table 22]

[0372] d. Follow-up of antitumor activity in the gastric PDX CTG-1234 model The ability of BisG1-FAPα-FERL / DR4-FERR to induce antitumor activity was evaluated in a low dose range in comparison with RG7386 in a murine gastric cancer-derived PDX model, CTG-1234. BisG1-b12-FERL / DR4-FERR was used as a negative control, and RG7386 was tested to compare the antitumor effects between BisG1-FAPα-FERL / DR4-FERR and RG7386. The experimental procedure was as described in section b of Example 12, with the following modifications: Mice were randomized into control and treatment groups at approximately 270 mm 2 The mean tumor volume (MTV) was established at the time of the study (Table 20).

[0373] [Table 23]

[0374] result BisG1-FAPα-FERL / DR4-FERR induced tumor regression and long-term tumor suppression in CTG-1234 tumors. Pairwise analysis of tumor volumes (TVs) on the final day (day 12) after all groups were completed showed that treatment with 2.0 and 0.5 mg / kg BisG1-FAPα-FERL / DR4-FERR significantly reduced TVs compared with the 2.0 mg / kg BisG1-b12-FERL / DR4-FERR control antibody and equimolar doses of RG7386 (Figures 10A-B, Table 21).

[0375] PFS was significantly prolonged in the group treated with 2.0 mg / kg BisG1-FAPα-FERL / DR4-FERR compared with the BisG1-b12-FERL / DR4-FERR control group and RG7386-treated mice (Figure 10C).

[0376] [Table 24]

[0377] [Example 13] Antitumor activity in a multi-organ metastatic mouse model The antitumor activity of BisG1-FAPα-FEAL / DR4-FEAR was investigated in a CRC PDO multi-organ metastasis mouse model using mouse fibroblasts as a source of FAPα. IgG1-b12-FEAL was used as a negative control.

[0378] Hub096 PDO (described in Example 11) was transduced using a lentivirus encoding luciferase linked to the green fluorescent protein (GFP) gene (PLV-luciferase-IRES-GFP lentiviral vector). Lentivirus encoding luciferase linked to GFP was produced in human embryonic kidney (HEK) 293T cells following a calcium phosphate transfection protocol.

[0379] On the day of transfection, HEK293T cells were detached with trypsin (Lonza, catalog no. BE02-007E), viability was confirmed with trypan blue (Fluka, catalog no. 93590), and plated onto 10 cm culture dishes (Greiner, catalog no. 664160) at a density of approximately 60% confluency the following day in 9 mL of DMEM high glucose medium (Sigma-Aldrich, catalog no. D6429) supplemented with 50 U / mL penicillin / streptomycin (Gibco, catalog no. 15070-063), 2 mM GlutaMAX (Gibco, catalog no. 35050-038), and 10% heat-inactivated FBS (Bodinco BV, ID 5067V20002). Cells were incubated at 37°C and 5% (vol / vol) CO2 for 24 hours to allow attachment to the plates. The next day, the medium was gently washed twice with PBS (Corning, Cat. No. 21-0310CVR) and fresh DMEM high glucose medium (supplemented with 50 U / mL penicillin / streptomycin, 2 mM GlutaMAX, and 10% heat-inactivated FBS) was added. 500 μL of 2× HEPES-buffered saline (HBS), pH 7.05, consisting of 280 mM sodium chloride (Riedel-de-Haen, catalog no. 31434), 1.5 mM sodium phosphate dibasic (Sigma, catalog no. S0876), 12 mM (d) glucose (Sigma, catalog no. G8270), 10 mM potassium chloride (Riedel-de-Haen, catalog no. 31248), and 50 mM HEPES (Sigma, catalog no. H3375) was dissolved in 50 μL of 3 M calcium chloride dihydrate (Riedel-de-Haen, catalog no. 31307), and 20 μg plasmid DNA (10 μg PLV-luciferase-IRES-GFP, 5 μg psPAX (Addgene) [plasmid #12260], and 5 μg pCMV-VSV-G [Addgene, plasmid # The transfection mix was mixed with 450 μL of PBS containing 10 ...The next day, HEK293T cells were washed twice with PBS and 6 mL of fresh DMEM high glucose medium supplemented with penicillin / streptomycin, GlutaMAX, and heat-inactivated 10% FBS was added to a 10 cm culture dish and further incubated overnight. The next day, Hub096 PDOs were dissociated using TrypLE Express Stable Trypsin-Like Enzyme (Gibco, catalog no. 12604021), and 1–2 million PDOs were seeded onto non-adherent 6-well plates (Corning, catalog no. 3471) in 6 mL of lentivirus-containing medium (collected from virus-producing HEK293T cell culture plates and filtered through a 0.22 μm polyethersulfone filter (Sarstedt BV, catalog no. 83.1826.001)) supplemented with 6 μg / mL polybrene (Sigma-Aldrich, catalog no. TR-1003), 0.5 mM N-acetylcysteine ​​(Sigma-Aldrich, catalog no. A9165), and 10 μM ROCK inhibitor Y-27632 (Abmole bioscience, catalog no. HY-10583). PDOs were incubated overnight at 37°C and 5% CO2. After 24 h of incubation, PDOs were collected in 15 mL tubes (Corning, catalog no. 430791), washed twice with PBS (Corning, catalog no. 21-031-CV), and resuspended in basement membrane extract (BME) matrix (Amsbio, catalog no. 3533-010-02) at a 2:1 ratio with PDO medium (see Example 11 for medium composition). PDOs were seeded in 150 μL droplets (containing approximately 2,500–5,000 cells per droplet), which were allowed to solidify at 37°C and 5% CO2. After solidification, 2 mL of PDO medium was added per well and the mixture was incubated at 37°C and 5% CO2. After at least two passages to allow cells to expand, GFP-positive PDOs were sorted using a fluorescence-activated cell sorting (FACS) Aria II (BD Biosciences) machine.

[0380] The day before transplantation, Hub096 PDOs were harvested, washed with PBS (Corning, catalog no. 21-031-CVR), and dissociated into single cells using TrypLE (Thermofisher, catalog no. 12604013). Cells were then washed, resuspended in PDO medium, and viability was measured with trypan blue. Cells were washed and resuspended in a pre-cooled solution of rat tail high-concentration type I collagen (Corning, catalog no. 354249) mixed at a 4:1 ratio with 5x neutralization buffer (1 g of AlphaMEM powder 5X (Life Technologies, catalog no. 12000-014); 5 mL of 1 M HEPES pH 7.5 (Lonza, catalog no. 17737E); 1 g of sodium bicarbonate (Sigma, catalog no. 31437)). A 10 μL droplet containing 300,000 single cells was added to a pre-warmed 6-well plate (Corning, catalog no. 3506). The plate was incubated (37°C, 5% CO) for 40-60 min to allow the droplets to solidify, after which 2 mL / well of PDO medium was added. The PDO was allowed to recover overnight at 37°C, 5% CO.

[0381] For cecal transplantation, male test mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ NSG® (Charles River Laboratories, strain #005557; 8-9 weeks old at the start of the experiment) were treated with a subcutaneous injection of carprofen (5 mg / kg, Rimady™) 30 minutes before surgery. To perform surgery, animals were sedated (isoflurane inhalation anesthesia: approximately 2% [vol / vol] isoflurane / O2 mixture), the cecum was exteriorized through a midline abdominal incision, and a drop of collagen containing luciferase-tagged PDO was surgically implanted into the cecal submucosa.

[0382] Mice were randomized into groups (n=9 / group). Treatment began 2 weeks after cecal transplantation and was administered once at weeks 2, 5, and 6, and twice weekly at weeks 3 and 4. On the day of treatment, mice were injected intraperitoneally with antibody (2 mg / kg in 200 μL of PBS).

[0383] Mice were monitored daily for clinical signs of disease and weighed weekly using a digital scale. Data including individual and mean gram weights and mean percent weight change relative to day 0 (%vD0) were recorded for each group. Animal deaths, if any, were recorded. Study toxicity endpoints were reported as follows: mean loss of %vD0 >20 and / or net weight loss >15% within 2 days, the discovery of mice moribund or displaying abnormal behavior and posture, and the appearance of a combination of clinical symptoms (large abdomen, ascites) that may indicate excessive tumor growth and metastasis.

[0384] Prior to study termination, mice were injected intraperitoneally with 100 μL (1.25 mg) of luciferin (VivoGlo™ Luciferin, In Vivo Grade, Promega, Catalog No. P1041) and euthanized 10 minutes later. Individual organs (cecum, peritoneal wall, liver, lungs, and brain) were collected and measured for tumor burden using ex vivo bioluminescence imaging (BioLI). Data were analyzed using GraphPad Prism 9, and BioLI measurements (cpm / cm) for organs from each mouse were calculated. 2 , Log 10 The mean (logarithmic scale) of the biomarkers was plotted for both treatment groups. Differences in BioLI were assessed by comparing treatment groups with the control group on log-transformed data using paired and unpaired non-parametric t-tests.

[0385] Additionally, DR4 activation was assessed by cleaved caspase-3 IHC staining on FFPE sections from cecum, peritoneal wall, and liver tissue. After dissection and BioLI measurement, cecum, peritoneal wall, and liver tissue were fixed in 4% (w / v) formaldehyde (Added Pharma, catalog number ROL.1642810) and embedded in Surgipath Paraplast paraffin (Leica Biosystems, catalog number #39602012) in laser biopsy green cassettes (FA-Tech Diagnostics Europe BV, catalog number #215-05-10LM) using a HistoCore Arcadia H (Leica Biosystems, catalog number #14039357258). Tissue blocks were cut into 4 μm serial sections parallel to the longitudinal axis of each tissue using a microtome (Leica Biosystems, catalog number #RM2255), transferred to the surface of a water bath (KLINIPATH, catalog number #WB28040), and mounted on X-tra Slides (Leica Biosystems, catalog number #3800203AE). Slides were then dried on a slide warmer (Adamas Instruments BV catalog #SW85). FFPE sections of cecum, peritoneal wall, and liver tissue were then deparaffinized with xylene (Klinipath, catalog #4055-9005) and rehydrated in serial dilutions of ethanol (Klinipath, catalog #4096-9005) and water. Endogenous peroxidase activity was blocked with 5% H2O2 (Merck, Catalog No. 1072091000) diluted in PBS (1.87 M NaCl (Merck, Catalog No. 1064041000), 0.28 M NaH2PO4 (Sigma-Aldrich, Catalog No. 7558-79-4), and 0.0366 M NaH2PO4 (Merck, Catalog No. 1063451000)) for 20 min at room temperature. Slides were incubated in boiling 10 mM citrate antigen retrieval buffer pH 6.0 (ThermoFisher Scientific, Catalog No. 36439) for 20 min.After cooling for 10 minutes, the slides were washed with 0.05% Tween 20 buffer (VWR, catalog no. M147-1L) diluted in PBS and incubated with cleaved caspase-3 (CCASP3) antibody (Cell Signaling Technology, catalog no. 9661, 1:300 in PBS supplemented with 1% BSA and 0.2% sodium azide) for 1 hour at room temperature. The slides were washed three times with Tween 20 buffer and incubated with BrightVision+ poly-HRP conjugated anti-rabbit IgG (Immunologic, catalog no. VWRKDPVR110HRP) for 30 minutes at room temperature. After three washes with PBS, the slides were incubated with 3,3'-diaminobenzidine (DAB) HO solution (0.03% DAB [Sigma-Aldrich, catalog number 91-95-2] and 0.03% HO [Merck, catalog number 7722-84-1] in 0.05 M Tris-HCl buffer pH 7.6 [Biosolve, catalog number 20092391]) at 37°C for 10 minutes. The slides were washed with water and stained with hematoxylin (Merck, catalog number HHS32; 1:4 in water) for 30 seconds at room temperature. After washing with water and then 96% ethanol, the slides were air-dried and coverslipped using ClearVue™ coverslippers (Thermo Scientific). Stained cecum, liver, and peritoneal wall slides were scanned at 40x magnification with a resolution of 0.25 μm / pixel using a NanoZoomer-XR digital slide scanner (Hamamatsu). QuPath software was used for automatic recognition of background, tissue (hematoxylin), and CCASP3-stained (DAB-positive) areas. Subsequently, each tumor area (excluding necrotic areas) was manually outlined and annotated. The percentage CCASP3-positive tumor area was determined using QuPath's trained pixel classifier.

[0386] result Ex vivo BioLI measurements of tumor burden in individual organs showed that, compared with the IgG1-b12-FEAL antibody, the BisG1-FAPα-FEAL / DR4-FEAR antibody significantly reduced tumor burden at the primary tumor site (cecum) and metastases in the brain, liver, peritoneal wall, and lungs using a paired t-test (Figures 11A-11E; Table 22). Using an unpaired t-test (Mann-Whitney), reductions in tumor burden were demonstrated at the primary tumor site (cecum) and metastatic sites, reaching statistical significance at metastatic sites including the brain, liver, peritoneal wall, and lungs (Table 22).

[0387] In mice treated with BisG1-FAPα-FEAL / DR4-FEAR, DR4 activation was observed in primary tumors and metastatic sites, as demonstrated by a significant increase in the percentage of tumor area that stained positive for cleaved caspase-3 on FFPE sections of cecum and peritoneal wall tissue compared with IgG1-b12-FEAL (Figure 11F, Table 23). Compared with cecum and peritoneal wall tissue, caspase-3 activity in the liver was low and not significantly increased by treatment with BisG1-FAPα-FEAL / DR4-FEAR.

[0388] [Table 25]

[0389] [Table 26]

[0390] In this study, treatment of NSG mice implanted with CRC-derived PDO with BisG1-FAPα-FEAL / DR4-FEAR resulted in potent antitumor activity against primary tumors and metastases compared with the negative control IgG1-b12-FEAL.

[0391] [Example 14] Evaluation of hepatotoxicity using human liver spheroids TRAIL-R agonists have been shown to induce hepatotoxicity in some patients in clinical studies. Therefore, we developed a tumor-specific agonist (BisG1-FAPα-FEAL / DR4-FEAR) that should reduce the risk of hepatotoxicity. Hepatotoxicity assays were performed to evaluate the potential of BisG1-FAPα-FEAL / DR4-FEAR, ABBV-621-Fc fusion, and RG7386 to induce hepatotoxicity in vitro. IgG1-b12-FEAR and IgG1-b12 were included as negative controls.

[0392] Drug-induced liver injury (DILI-Bio) safety assessment was performed using Human 3D InSight™ Human Liver Microtissues (InSphero, catalog number MT-02-302-04), which are liver spheroids composed of primary human hepatocytes and non-parenchymal liver cell types such as Kupffer cells and hepatic endothelial cells. Two experiments were performed, and the viability of the liver spheroids was determined by measuring lactate dehydrogenase (LDH) release, an indicator of plasma membrane damage, on day 4 and intracellular adenosine triphosphate (ATP) levels, an indicator of metabolically active cells, on days 6 and 7.

[0393] The first experiment was performed according to InSphero's internal technical operating procedure (TOP). Briefly, antibody dilutions (final concentrations of 2 μg / mL, 10 μg / mL, and 50 μg / mL using TOX microtissue culture medium (Insphero, catalog number CS-07-001-01)) were added to 96-well Insphero Plates (catalog number MT-02-302-04). Plates were incubated at 37°C and 5% CO2. Extracellular LDH release was measured on day 4 of treatment using a bioluminescent LDH release toxicity assay kit (Promega, catalog number J2380), and intracellular ATP content was measured on day 6 of treatment using a CellTiter-Glo 2.0 cell viability assay (Promega, catalog number G9243).

[0394] The second experiment was performed as follows. The Insphero Plates were centrifuged to ensure that the microtissues were at the bottom of the wells, after which antibody dilutions (same as in the first experiment, diluted in TOX medium) were added. Plates were incubated at 37°C and 5% CO for up to 7 days. For LDH measurements, supernatants were collected after 4 days and used with a bioluminescent LDH release toxicity assay (Promega, catalog no. J2381) according to the manufacturer's protocol. For ATP measurements, supernatants were removed after 7 days and 50 μL / well of CellTiter-Glo 3D (Promega, catalog no. G9681) reagent (diluted 1:1 with PBS (Hyclone GE Healthcare, catalog no. SH3A3830.03)) was added. After mixing, the entire volume was transferred to a 96-well white OptiPlate (Perkin Elmer, catalog no. 6005299). After 30 minutes of incubation at room temperature, protected from light, bioluminescence was measured using EnVision (Perkin Elmer). For both experiments, data were analyzed using Microsoft Excel and GraphPad Prism. Data shown are the mean ± SEM of four technical replicates.

[0395] result Treatment of liver spheroids with the ABBV-621-Fc fusion induced toxicity in hepatocytes, as indicated by increased release of LDH into the culture supernatant (Fig. 12A). Treatment with higher doses of RG7386 also induced significant LDH release (Fig. 12C). In contrast, BisG1-FAPα-FEAL / DR4-FEAR did not induce toxicity in this liver spheroid model, as the level of LDH in the culture supernatant was below the LLOD (dashed line), similar to the negative control IgG1-b12-FEAR or IgG1-b12 (Fig. 12A / C).

[0396] Cellular ATP levels in liver spheroids treated with low doses of ABBV-621-Fc fusion and RG7386 were similar to those treated with BisG1-FAPα-FEAL / DR4-FEAR or control IgG1-b12-FEAR antibody (Figure 12B). However, a decrease in cellular ATP levels was observed with the highest doses of ABBV-621-Fc fusion and RG7386 (Figure 12B / Figure 12D), indicating a loss of hepatocyte survival.

[0397] In summary, the BisG1-FAPα-FEAL / DR4-FEAR antibody did not induce liver spheroid toxicity (survival indices comparable to those of the negative control IgG1-b12-FEAR / IgG1-b12). In contrast, the ABBV-621-Fc fusion and RG7386 showed dose-dependent toxicity.

[0398] [Example 15] Cytotoxicity in co-cultures with reduced availability of FAPα To investigate the relationship between FAPα expression levels and BisG1-FAPα-FERL / DR4-FERR-induced cytotoxicity, we investigated the role of the tumor cell to FAPα-expressing fibroblast ratio on BisG1-FAPα-FERL / DR4-FERR efficacy.

[0399] Cytotoxicity in cocultures of tumor cells DLD-1 and MDA-MB-231 with different amounts of NIH / 3T3-FAPα cells was assessed using the CellTiter Glo assay. DR4-expressing tumor cells (MDA-MB-231 and DLD-1) were harvested as described in Example 3. NIH-3T3-FAPα cells were harvested as described in Example 10. Tumor cells and fibroblasts were seeded into CELLSTAR® flat-bottom 96-well plates (Greiner Bio, catalog no. #655180) at the indicated ratios (6,600 tumor cells and various numbers of fibroblasts [100–3,300 cells / well in 2-fold increments]) and incubated at 37°C and 5% CO2 for 4 hours to allow cells to adhere to the plates. Control wells were prepared in parallel using monocultures of tumor cells or NIH / 3T3-FAPα cells. The supernatant was then removed and the cells were resuspended in RPMI 1640 (Gibco, Cat. No. A1049101) containing 10% DBSI (Gibco, Cat. No. 10371029) at a concentration series of antibodies (6.91 × 10 -6 The tumor cells were incubated with 100 μg / mL of IgG4-dependent antibody (~14.5 μg / mL, 8-fold increments) at 37°C and 5% CO2 for 72 hours. Cultured cell viability was assessed using the homogenous CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Catalog No. G7571), as described in Example 10, Section a, below. The percentage of remaining viable tumor cells was calculated, and the data was processed with GraphPad Prism software to generate fitted cell viability curves (nonlinear regression analysis with four-parameter logistic curve fitting). The graph shows the percentage of viable tumor cells plotted against antibody concentration.

[0400] Cytotoxicity was further evaluated in cocultures of PDO with CAFs transduced with FAPα shRNA or non-targeting shRNA. Different FAPα shRNA clones were generated using a calcium phosphatase transfection-based protocol for lentiviral vectors in HEK293T cells and used to transduce CAFs. Lentiviral production in HEK293T cells was performed as described in Example 13, with the following modifications. The transfection mix contained the following vectors: 15 μg of MISSION pLKO.1-puro base plasmid (Sigma-Aldrich, catalog number #Scr:SHC016, #34:TRCN 0000006802), 7.5 μg of psPAX2 (Addgene, plasmid #12260), and 7.5 μg of pCMV-VSV-G (Addgene, plasmid #8454). After collecting the lentivirus-containing medium, the transduction medium was filtered using a 0.22 μm polyethersulfone filter (Sarstedt BV catalog 83.1826.001) and supplemented with 3 μg / mL polybrene (Sigma-Aldrich catalog #H9268). CAFs were collected, counted, and seeded onto collagen-coated 6-well plates as described in Example 11 in the lentivirus-containing filtered medium (2.5 mL / well) and incubated at 37°C and 5% CO2 for growth (2.5 mL / well). Transduced CAFs were collected and resuspended in medium supplemented with 0.5 μg / mL puromycin dihydrochloride (Santa Cruz Biotechnology, catalog #sc-108071A). To continue puromycin selection, the medium was replaced with fresh medium and puromycin every 2–3 days. Subsequently, CellTiter-Glo survival assays were performed using PDO and CAFs as described in Example 11.

[0401] result To investigate the relationship between FAPα expression levels and BisG1-FAPα-FERL / DR4-FERR-induced cytotoxicity, we investigated the role of the tumor cell to FAPα-expressing fibroblast ratio on cytotoxicity. DLD-1 or MDA-MB-231 tumor cell lines were cultured with different amounts of NIH-3T3-FAPα cells (tumor cell to NIH-3T3-FAPα ratios of 2:1, 4:1, 8:1, and 64:1). An increase in the tumor cell to fibroblast ratio (e.g., a decrease in NIH / 3T3-FAPα cells) reduced BisG1-FAPα-FERL / DR4-FERR-mediated cytotoxicity (Figures 13A-B).

[0402] Furthermore, the effects of BisG1-FAPα-FERL / DR4-FERR were studied in cocultures with fibroblasts with different levels of FAPα expression. CAFs were transduced with FAPα shRNA (CAF#34) or non-targeting shRNA (CAF#Scr). In cocultures with CAFs (CAF#34) engineered to express lower levels of surface FAPα from four PDO lines, the maximal BisG1-FAPα-FERL / DR4-FERR-mediated cytotoxic effect was unaffected in one PDO line, slightly reduced in two PDO lines, and completely lost in one PDO line (Figures 14A-D). In all PDO lines, a bell-shaped concentration-response relationship was observed in cocultures with engineered CAFs expressing reduced levels of FAPα. In conclusion, cytotoxicity mediated by BisG1-FAPα-FERL / DR4-FERR depends on the density and expression level of FAPα.

[0403] [Example 16] Cytotoxicity and DR4 transactivation in the presence of soluble FAPα Given that FAPα protein can be released from the cell membrane and is found in human plasma (Xin et al., Front Oncol 2021 11:648187), the effect of soluble FAPα on BisG1-FAPα-FERL / DR4-FERR-mediated cytotoxicity was evaluated. The cytotoxicity of BisG1-FAPα-FERL / DR4-FERR and the control antibody BisG1-b12-FERL / DR4-FERR was evaluated in monocultures of DR4-expressing DLD-1 and MDA-MB-231 tumor cells as described in Example 15, with the following modifications: tumor cells were grown in monoculture, and where indicated, recombinant human FAPα (BioLegend®, Catalog No. #76908, fixed concentration of 29.4 nM) was added simultaneously with the antibody samples.

[0404] result In tumor cell monocultures (DLD-1 and MDA-MB-231), addition of recombinant human FAPα at 29.4 nM, a concentration equivalent to 10-fold the maximum concentration reported in human plasma, did not induce BisG1-FAPα-FERL / DR4-FERR-mediated cytotoxicity (Figures 15A-B). Thus, BisG1-FAPα-FERL / DR4-FERR-induced transactivation-mediated cell death required the presence of cell surface-expressed FAPα.

[0405] [Example 17] Effect on FAPα enzyme activity in vitro The effect of BisG1-FAPα-FERL / DR4-FERR on FAPα dipeptidyl peptidase (DPP) activity was examined using a fluorogenic assay in which a fluorogenic dipeptidyl peptidase substrate was incubated with samples containing soluble FAPα.

[0406] As a readout of FAPα enzyme activity, the fluorescent signal generated by the release of 7-amino-4-methylcoumarin (AMC) from the dipeptidyl peptidase substrate can be measured using a fluorescence reader. FAPα dipeptidyl peptidase activity was tested using the FAP Fluorogenic Assay Kit (BPS Bioscience, catalog number #80210) according to the manufacturer's instructions. The fluorogenic dipeptidyl peptidase substrate was incubated in the presence of FAPα in the presence of BisG1-FAPα-FERL / DR4-FERR, the non-binding control antibody BisG1-b12-FERL / b12-FERR, or the chemical dipeptidyl peptidase inhibitor Talabostat (Cayman Chemical, catalog number CAYM290075) as a positive control for enzyme inhibition. The fluorescent signal resulting from the ...

Claims

1. at least (i) a FAPα-binding region comprising a first heavy chain variable region and a first light chain variable region; and (ii) a DR4-binding region comprising a second heavy chain variable region and a second light chain variable region; A multispecific antibody comprising:

2. 2. The multispecific antibody of claim 1, wherein the FAPα-binding region comprises a heavy chain variable region (VH) comprising three complementarity-determining regions, CDR1, CDR2 and CDR3, present within the amino acid sequence set forth in SEQ ID NO:

13.

3. 3. The multispecific antibody according to claim 1 , wherein the FAPα-binding region comprises a light chain variable region (VL) comprising three complementarity-determining regions, CDR1, CDR2 and CDR3, present within the amino acid sequence shown in SEQ ID NO:

14.

4. 4. The multispecific antibody according to claim 1 , wherein the FAPα-binding region comprises a heavy chain variable region (VH) comprising the three complementarity-determining regions CDR1, CDR2 and CDR3 present in the amino acid sequence shown in SEQ ID NO: 13, and a light chain variable region (VL) comprising the three complementarity-determining regions CDR1, CDR2 and CDR3 present in the amino acid sequence shown in SEQ ID NO:

14.

5. 5. The multispecific antibody of claim 1 , wherein the FAPα binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 1, 2 and 3, respectively.

6. 6. The multispecific antibody of claim 1 , wherein the FAPα binding region comprises a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 4, 5 and 6, respectively.

7. 7. The multispecific antibody of claim 1 , wherein the FAPα binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 1, 2 and 3, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 4, 5 and 6, respectively.

8. 8. The multispecific antibody of claim 1 , wherein the VH sequence of the FAPα-binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:

13.

9. 9. The multispecific antibody of claim 1, wherein the VL sequence of the FAPα-binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:

14.

10. 10. The multispecific antibody of claim 1, wherein the VH sequence of the FAPα-binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 13, and the VL sequence of the FAPα-binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:

14.

11. 11. The multispecific antibody of claim 1 , wherein the framework regions of the VH sequence of the FAPα-binding region have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the framework regions of the amino acid sequence set forth in SEQ ID NO:

13.

12. 12. The multispecific antibody of claim 1 , wherein the framework regions of the VL sequence of the FAPα-binding region have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the framework regions of the amino acid sequence set forth in SEQ ID NO:

14.

13. 13. The multispecific antibody of claim 1, wherein the framework regions of the VH sequence of the FAPα-binding region have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the framework regions of the amino acid sequence set forth in SEQ ID NO: 13, and the framework regions of the VL sequence of the FAPα-binding region have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the framework regions of the amino acid sequence set forth in SEQ ID NO:

14.

14. 14. The multispecific antibody of any one of claims 1 to 13, wherein the VH sequence of said FAPα binding region differs from SEQ ID NO: 13 by at most 10 substitutions, such as at most 9 substitutions, for example by at most 8 substitutions, such as at most 7 substitutions, for example by at most 6 substitutions, such as at most 5 substitutions, for example by at most 4 substitutions, such as at most 3 substitutions, for example by at most 2 substitutions, such as at most 1 substitution.

15. 15. The multispecific antibody of any one of claims 1 to 14, wherein the VL sequence of the FAPα binding region differs from SEQ ID NO: 14 by at most 10 substitutions, such as at most 9 substitutions, for example by at most 8 substitutions, such as at most 7 substitutions, for example by at most 6 substitutions, such as at most 5 substitutions, for example by at most 4 substitutions, such as at most 3 substitutions, for example by at most 2 substitutions, such as at most 1 substitution.

16. The multispecific antibody of any one of claims 1 to 15, wherein the VH and VL sequences of the FAPα binding region differ only in the framework regions.

17. 17. The multispecific antibody of any one of claims 1 to 16, wherein the VH sequence of the FAPα binding region comprises, consists essentially of or consists of the VH sequence as shown in SEQ ID NO:

13.

18. 18. The multispecific antibody of any one of claims 1 to 17, wherein the VL sequence of the FAPα binding region comprises, consists essentially of, or consists of the VL sequence shown in SEQ ID NO:

14.

19. 19. The multispecific antibody according to any one of claims 1 to 18, wherein the VH and VL sequences of the FAPα binding region comprise, consist essentially of, or consist of the VH sequence set forth in SEQ ID NO: 13 and the VL sequence set forth in SEQ ID NO:

14.

20. 20. The multispecific antibody of any one of claims 1 to 19, wherein the FAPα is human FAPα, such as the mature polypeptide of SEQ ID NO: 33 or the soluble FAPα of SEQ ID NO: 34; the FAPα is mouse FAPα, such as the mature polypeptide of SEQ ID NO: 35; the FAPα is rat FAPα, such as the mature polypeptide of SEQ ID NO: 36; the FAPα is canine FAPα, such as the mature polypeptide of SEQ ID NO: 37; the FAPα is porcine FAPα, such as the mature polypeptide of SEQ ID NO: 38; or the FAPα is cynomolgus FAPα, such as the mature polypeptide of SEQ ID NO:

39.

21. 21. The multispecific antibody according to any one of claims 1 to 20, wherein the FAPα is a human FAPα, such as the mature polypeptide of SEQ ID NO: 33 or the soluble FAPα of SEQ ID NO: 34; or a cynomolgus FAPα, such as the mature polypeptide of SEQ ID NO: 39, preferably a human FAPα, such as the mature polypeptide of SEQ ID NO:

33.

22. 22. The multispecific antibody of any of claims 1 to 21, wherein the DR4 binding region comprises a heavy chain variable region (VH) comprising three complementarity determining regions, CDR1, CDR2 and CDR3, present within the amino acid sequence set forth in SEQ ID NO:

15.

23. 23. The multispecific antibody of any of claims 1 to 22, wherein the DR4 binding region comprises a light chain variable region (VL) comprising three complementarity determining regions, CDR1, CDR2 and CDR3, present within the amino acid sequence set forth in SEQ ID NO:

16.

24. 24. The multispecific antibody according to any of claims 1 to 23, wherein the DR4 binding region comprises a heavy chain variable region (VH) comprising the three complementarity determining regions CDR1, CDR2 and CDR3 present in the amino acid sequence shown in SEQ ID NO: 15, and a light chain variable region (VL) comprising the three complementarity determining regions CDR1, CDR2 and CDR3 present in the amino acid sequence shown in SEQ ID NO:

16.

25. 25. The multispecific antibody of any one of claims 1 to 24, wherein the DR4 binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 8 and 9, respectively.

26. 26. The multispecific antibody of any one of claims 1 to 25, wherein the DR4 binding region comprises a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, 11 and 12, respectively.

27. 27. The multispecific antibody of any one of claims 1 to 26, wherein the DR4 binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 8 and 9, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, 11 and 12, respectively.

28. 28. The multispecific antibody of any of claims 1 to 27, wherein the VH sequence of the DR4 binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:

15.

29. 29. The multispecific antibody of any of claims 1 to 28, wherein the VL sequence of the DR4 binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:

16.

30. 30. The multispecific antibody of any of claims 1 to 29, wherein the VH sequence of the DR4 binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 15, and the VL sequence of the DR4 binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:

16.

31. 31. The multispecific antibody of any of claims 1 to 30, wherein the framework regions of the VH sequence of the DR4 binding region have at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity with the framework regions of the amino acid sequence set forth in SEQ ID NO:

15.

32. 32. The multispecific antibody of any of claims 1 to 31, wherein the framework regions of the VL sequence of the DR4 binding region have at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity with the framework regions of the amino acid sequence set forth in SEQ ID NO:

16.

33. 33. The multispecific antibody of any of claims 1 to 32, wherein the framework regions of the VH sequence of the DR4 binding region have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the framework regions of the amino acid sequence set forth in SEQ ID NO: 15, and the framework regions of the VL sequence of the DR4 binding region have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the framework regions of the amino acid sequence set forth in SEQ ID NO:

16.

34. 34. The multispecific antibody of any one of claims 1 to 33, wherein the VH sequence of said DR4 binding region differs from SEQ ID NO: 15 by at most 10 substitutions, such as at most 9 substitutions, for example by at most 8 substitutions, such as at most 7 substitutions, for example by at most 6 substitutions, such as at most 5 substitutions, for example by at most 4 substitutions, such as at most 3 substitutions, for example by at most 2 substitutions, such as at most 1 substitution.

35. 35. The multispecific antibody of any one of claims 1 to 34, wherein the VL sequence of said DR4 binding region differs from SEQ ID NO: 16 by at most 10 substitutions, such as at most 9 substitutions, for example by at most 8 substitutions, such as at most 7 substitutions, for example by at most 6 substitutions, such as at most 5 substitutions, for example by at most 4 substitutions, such as at most 3 substitutions, for example by at most 2 substitutions, such as at most 1 substitution.

36. 36. The multispecific antibody of any one of claims 1 to 35, wherein the VH and VL sequences of the DR4 binding domains differ only in the framework regions.

37. 37. The multispecific antibody of any of claims 1 to 36, wherein the VH sequence of the DR4 binding region comprises, consists essentially of, or consists of the VH sequence shown in SEQ ID NO:

15.

38. 38. The multispecific antibody of any of claims 1 to 37, wherein the VL sequence of the DR4 binding region comprises, consists essentially of, or consists of the VL sequence shown in SEQ ID NO:

16.

39. 39. The multispecific antibody according to any of claims 1 to 38, wherein the VH and VL sequences of the DR4 binding region comprise, consist essentially of, or consist of the VH sequence set forth in SEQ ID NO: 15 and the VL sequence set forth in SEQ ID NO:

16.

40. 40. The multispecific antibody of any one of claims 1 to 39, wherein DR4 is human DR4, such as the mature polypeptide of SEQ ID NO: 68, or cynomolgus DR4, such as the mature polypeptide of SEQ ID NO:

69.

41. The multispecific antibody of any one of claims 1 to 40, wherein DR4 is human DR4, such as the mature polypeptide of SEQ ID NO:

68.

42. The multispecific antibody of any one of claims 1 to 41, which is a bispecific antibody.

43. K of FAPα binding to the FAPα binding region Ｄ but, or less than 1000 pM, such as 900 pM or less, for example 800 pM or less, for example 700 pM or less, such as 600 pM or less, for example 500 pM or less, such as 400 pM or less, for example 300 pM or less, such as 200 pM or less, for example 100 pM or less, such as 90 pM or less, for example 80 pM or less, for example 70 pM or less, such as 60 pM or less, for example 50 pM or less, for example 40 pM or less, such as 30 pM or less, or 43. The multispecific antibody according to any of claims 1 to 42, wherein the antibody is in the range of 0.1 pM to 1000 pM, such as 0.5-900 pM, for example 1 pM to 800 pM, such as 2 pM to 700 pM, for example 3 pM to 600 pM, such as 4 pM to 500 pM, for example 5 pM to 400 pM, such as 6 pM to 300 pM, for example 7 pM to 200 pM, such as 8 pM to 100 pM, for example 9 pM to 75 pM, such as 10 pM to 50 pM.

44. K of DR4 that binds to the DR4 binding region Ｄ but, is 100 nM or less, such as 90 nM or less, for example 80 nM or less, for example 70 nM or less, such as 60 nM or less, for example 50 nM or less, such as 40 nM or less, for example 30 nM or less, such as 20 nM or less, for example 10 nM or less, such as 9 nM or less, for example 8 nM or less, such as 7 nM or less, for example 6 nM or less, such as 5 nM or less, for example 4 nM or less, for example 3 nM or less, such as 2 nM or less, for example 1 nM or less, such as 0.5 nM or less; 44. The multispecific antibody of any of claims 1 to 43, wherein the antibody is in the range of 0.01 nM to 10 nM, such as 0.02 nM-9 nM, for example 0.03 nM-8 nM, such as 0.04 nM to 7 nM, for example 0.05 nM to 6 nM, such as 0.075 nM to 5 nM, for example 0.1 nM to 4 nM, such as 0.15 nM to 3 nM, for example 0.2 nM to 2 nM, such as 0.25 nM to 1 nM, for example 0.3 nM to 0.75 nM.

45. K of FAPα that binds to the FAPα binding region Ｄ is 100 pM or less, and the K Ｄ The multispecific antibody of any one of claims 1 to 44, wherein the antibody has a specific activity of 1 nM or less.

46. The antibody (i) a FAPα binding region comprising a first heavy chain variable region and a first light chain variable region, wherein the heavy chain variable region (VH) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 1, 2 and 3, respectively, and the light chain variable region (VL) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 4, 5 and 6, respectively; (ii) a DR4 binding region comprising a second heavy chain variable region and a second light chain variable region, wherein the heavy chain variable region (VH) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 7, 8, and 9, respectively, and the light chain variable region (VL) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 10, 11, and 12, respectively; and 46. ​​The multispecific antibody of any one of claims 1 to 45, comprising:

47. The antibody (i) a FAPα binding region comprising, consisting essentially of, or consisting of the VH sequence set forth in SEQ ID NO: 13 and the VL sequence set forth in SEQ ID NO: 14; (ii) a DR4 binding region comprising, consisting essentially of, or consisting of the VH sequence set forth in SEQ ID NO: 15 and the VL sequence set forth in SEQ ID NO: 16; 47. The multispecific antibody of any one of claims 1 to 46, comprising:

48. (i) the antibody comprises a first heavy chain and a second heavy chain, wherein the first heavy chain comprises the VH region of the FAPα-binding region, and the second heavy chain comprises the VH region of the DR4-binding region; (ii) the first heavy chain and the second heavy chain each comprise at least a hinge region, a CH2 region, and a CH3 region; and (iii) in the first heavy chain, at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (in a human IgG1 heavy chain according to Eu numbering) is substituted, and in the second heavy chain, at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (in a human IgG1 heavy chain according to Eu numbering) is substituted. A multispecific antibody according to any one of claims 1 to 47.

49. 49. The multispecific antibody of any of claims 1 to 48, wherein the first heavy chain and the second heavy chain are not substituted at the same positions.

50. (i) the amino acid at the position corresponding to F405 (in a human IgG1 heavy chain according to Eu numbering) is L in the first heavy chain, and the amino acid at the position corresponding to K409 (in a human IgG1 heavy chain according to Eu numbering) is R in the second heavy chain; or (ii) the amino acid at the position corresponding to K409 (in a human IgG1 heavy chain according to Eu numbering) is R in the first heavy chain, and the amino acid at the position corresponding to F405 (in a human IgG1 heavy chain according to Eu numbering) is L in the second heavy chain; A multispecific antibody according to any one of claims 1 to 49.

51. 51. The multispecific antibody of any one of claims 1 to 50, wherein the antibody comprises a first heavy chain and a second heavy chain, and one or both heavy chains have been modified so as to reduce the extent to which the antibody induces Fc-mediated effector function compared to an identical antibody except for comprising unmodified first and second heavy chains.

52. 52. The multispecific antibody of any of claims 1 to 51, wherein said antibody induces Fc-mediated effector function to less than 95%, such as less than 90%, such as less than 85%, such as less than 80%, for example less than 75%, such as less than 70%, for example less than 65%, such as less than 60%, for example less than 55%, such as less than 50% of an antibody that is identical except for comprising unmodified first and second heavy chains.

53. 53. The multispecific antibody of any one of claims 1 to 52, wherein the antibody comprises a first heavy chain and a second heavy chain, wherein in at least one of the first and second heavy chains, one or more amino acids at positions corresponding to positions L234, L235, G236, D265, N297, and P331 in a human IgG1 heavy chain according to Eu numbering are not L, L, G, D, N, and P, respectively.

54. 54. The multispecific antibody of any of claims 1 to 53, wherein the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to Eu numbering are F and E, respectively, in the first and / or second heavy chain.

55. the antibody is a bispecific antibody comprising a first heavy chain and a second heavy chain, and wherein the positions in both the first heavy chain and the second heavy chain that correspond to positions L234 and L235 in a human IgG1 heavy chain according to Eu numbering are F and E, respectively, and wherein (i) the position in the first heavy chain corresponding to F405 in a human IgG1 heavy chain according to Eu numbering is L, and the position in the second heavy chain corresponding to K409 in a human IgG1 heavy chain according to Eu numbering is R; or (ii) the position in the first heavy chain corresponding to K409 in a human IgG1 heavy chain according to Eu numbering is R, and the position in the second heavy chain corresponding to F405 in a human IgG1 heavy chain according to Eu numbering is L; A multispecific antibody according to any one of claims 1 to 54.

56. 56. The multispecific antibody of any of claims 1 to 55, wherein the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain according to Eu numbering are F, E and A, respectively, in the first and / or second heavy chain.

57. the antibody is a bispecific antibody comprising a first heavy chain and a second heavy chain, and wherein the positions in both the first heavy chain and the second heavy chain corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to Eu numbering are F, E, and A, respectively, and wherein (i) the position in the first heavy chain corresponding to F405 in a human IgG1 heavy chain according to Eu numbering is L, and the position in the second heavy chain corresponding to K409 in a human IgG1 heavy chain according to Eu numbering is R; or (ii) the position in the first heavy chain corresponding to K409 in a human IgG1 heavy chain according to Eu numbering is R, and the position in the second heavy chain corresponding to F405 in a human IgG1 heavy chain according to Eu numbering is L; A multispecific antibody according to any one of claims 1 to 56.

58. 58. The multispecific antibody of any of claims 1 to 57, wherein the positions corresponding to positions L234, L235 and G236 in a human IgG1 heavy chain according to Eu numbering are F, E and R, respectively, in the first and / or second heavy chain.

59. the antibody is a bispecific antibody comprising a first heavy chain and a second heavy chain, and wherein the positions in both the first heavy chain and the second heavy chain corresponding to positions L234, L235, and G236 in a human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively, and wherein (i) the position in the first heavy chain corresponding to F405 in a human IgG1 heavy chain according to Eu numbering is L, and the position in the second heavy chain corresponding to K409 in a human IgG1 heavy chain according to Eu numbering is R; or (ii) the position in the first heavy chain corresponding to K409 in a human IgG1 heavy chain according to Eu numbering is R, and the position in the second heavy chain corresponding to F405 in a human IgG1 heavy chain according to Eu numbering is L; A multispecific antibody according to any one of claims 1 to 58.

60. 60. The multispecific antibody of any of claims 1 to 59, wherein one of the first and second heavy chains comprises substitutions of amino acids corresponding to amino acids at positions L234, L235 and G236 with F, E and R, respectively, and the other heavy chain comprises substitutions of amino acids corresponding to amino acids at positions L234, L235 and D265 with F, E and A, respectively, wherein said amino acid positions are as defined in Eu numbering.

61. the antibody is a bispecific antibody comprising a first heavy chain and a second heavy chain, and wherein one heavy chain comprises substitutions of amino acids corresponding to amino acids at positions L234, L235 and G236 with F, E and R, respectively, and the other heavy chain comprises substitutions of amino acids corresponding to amino acids at positions L234, L235 and D265 with F, E and A, respectively, and wherein (i) the position in the first heavy chain corresponding to F405 in a human IgG1 heavy chain according to Eu numbering is L, and the position in the second heavy chain corresponding to K409 in a human IgG1 heavy chain according to Eu numbering is R; or (ii) the position in the first heavy chain corresponding to K409 in a human IgG1 heavy chain according to Eu numbering is R, and the position in the second heavy chain corresponding to F405 in a human IgG1 heavy chain according to Eu numbering is L; A multispecific antibody according to any one of claims 1 to 60.

62. The multispecific antibody of any one of claims 1 to 61, which is an IgG1 antibody.

63. 63. The multispecific antibody of any one of claims 1 to 62, which is a full-length antibody.

64. 64. The multispecific antibody of any one of claims 1 to 63, which is a full-length IgG1 antibody.

65. 65. The multispecific antibody of any one of claims 1 to 64, comprising a kappa (κ) and / or a lambda (λ) light chain.

66. 66. The multispecific antibody of any one of claims 1 to 65, wherein the antibody comprises a heavy chain and a kappa (κ) light chain comprising a FAPα binding region, and a heavy chain and a lambda (λ) light chain comprising a DR4 binding region.

67. 67. The multispecific antibody of any of claims 1 to 66, wherein the DR4 binding region is comprised in a heavy chain and a light chain, the heavy chain comprising the VH region and an IgG1 heavy chain constant region, and the light chain comprising the VL region and a lambda light chain constant region, and wherein the FAPα binding region is comprised in a heavy chain and a light chain, the heavy chain comprising the VH region and an IgG1 heavy chain constant region, and the light chain comprising the VL region and a kappa light chain constant region.

68. 68. The multispecific antibody of any of claims 1 to 67, wherein one IgG1 heavy chain constant region is as defined in SEQ ID NO: 26 and the other is as defined in SEQ ID NO: 70, and wherein the kappa light chain constant region is as defined in SEQ ID NO: 27 and the lambda light chain constant region is as defined in SEQ ID NO:

28.

69. 69. The multispecific antibody of any one of claims 1 to 68, wherein the first and second Fc regions comprise the sequence of SEQ ID NO: 21 (IgG1m(f)), except for the specified mutations.

70. 70. The multispecific antibody of any one of claims 1 to 69, which is an antibody fragment.

71. the antibody, which may be a bispecific antibody, (i) a first heavy chain and a first light chain comprising a FAPα-binding region, wherein the FAPα-binding region comprises a first heavy chain variable region and a first light chain variable region, wherein the first heavy chain variable region (VH) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 1, 2 and 3, respectively, and the first light chain variable region (VL) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 4, 5 and 6, respectively; (ii) a second heavy chain and a second light chain comprising a DR4 binding region, wherein the DR4 binding region comprises a second heavy chain variable region and a second light chain variable region, wherein the second heavy chain variable region (VH) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 7, 8, and 9, respectively, and the second light chain variable region (VL) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 10, 11, and 12, respectively; and Including, (iii) wherein the positions in both the first heavy chain and the second heavy chain corresponding to positions L234, L235, and G236 in a human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively; and (iv) wherein the position in said first heavy chain corresponding to position F405 in a human IgG1 heavy chain according to Eu numbering is L, and the position in said second heavy chain corresponding to K409 in a human IgG1 heavy chain according to Eu numbering is R. A multispecific antibody according to any one of claims 1 to 70.

72. the antibody, which may be a bispecific antibody, (i) the FAPα heavy chain sequence set forth in SEQ ID NO: 17 and the FAPα light chain sequence set forth in SEQ ID NO: 18, and (ii) the DR4 heavy chain sequence shown in SEQ ID NO: 19 and the DR4 light chain sequence shown in SEQ ID NO: 20 72. The multispecific antibody of any of claims 1 to 71, comprising:

73. a first heavy chain and a first light chain linked via a disulfide bridge to form a first binding region that binds to FAPα, and a second heavy chain and a second light chain linked via a disulfide bridge to form a second binding region that binds to DR4, wherein: i) the first heavy chain comprises the sequence set forth in SEQ ID NO: 17 and the first light chain comprises the sequence set forth in SEQ ID NO: 18; and ii) the second heavy chain comprises the sequence set forth in SEQ ID NO: 19 and the second light chain comprises the sequence set forth in SEQ ID NO: 20; A multispecific antibody according to any one of claims 1 to 72.

74. 74. The multispecific antibody of any of claims 1 to 73, which is a bispecific, bivalent antibody having monovalent binding to FAPα and monovalent binding to DR4.

75. A nucleic acid construct or combination of nucleic acid constructs encoding an antibody according to any one of claims 1 to 74.

76. 76. The nucleic acid construct or combination of nucleic acid constructs of claim 75, wherein the combination of nucleic acid constructs comprises a first construct encoding the first heavy chain, a second construct encoding the second heavy chain, a third construct encoding the first light chain, and a fourth construct encoding the second light chain.

77. 76. The nucleic acid construct or combination of nucleic acid constructs of claim 75, wherein the combination of nucleic acid constructs comprises a first construct encoding the first heavy chain and the first light chain, and a second construct encoding the second heavy chain and the second light chain.

78. 78. An expression vector, or a combination of expression vectors, comprising one or more nucleic acid constructs according to any one of claims 75 to 77.

79. A composition comprising a nucleic acid construct or a combination of nucleic acid constructs according to any one of claims 75 to 77.

80. A delivery vehicle comprising one or more nucleic acid constructs according to any one of claims 75 to 77.

81. 81. The delivery vehicle of claim 80, wherein the delivery vehicle is a particle.

82. 82. The delivery vehicle of claim 81, wherein the particle is a lipid nanoparticle.

83. 83. The delivery vehicle of claim 82, wherein the lipid nanoparticle comprises a lipid, an ionizable amino lipid, a PEG-lipid, cholesterol, or any combination thereof.

84. 75. A recombinant host cell capable of producing an antibody according to any one of claims 1 to 74, said host cell comprising one or more nucleic acid constructs encoding an antibody as defined in any one of claims 1 to 74.

85. 85. The recombinant host cell of claim 84, which is a CHO cell.

86. 75. A pharmaceutical composition comprising a multispecific antibody as defined in any one of claims 1 to 74 and a pharmaceutically acceptable carrier.

87. A multispecific antibody according to any one of claims 1 to 74, one or more nucleic acid constructs according to any one of claims 75 to 77, a delivery vehicle according to any one of claims 80 to 83 or a pharmaceutical composition according to claim 86 for use as a medicament.

88. A multispecific antibody according to any of claims 1 to 74, one or more nucleic acid constructs according to any of claims 75 to 77, a delivery vehicle according to any of claims 80 to 83 or a pharmaceutical composition according to claim 86 for use in the treatment of cancer.

89. 89. The multispecific antibody, nucleic acid construct(s), delivery vehicle or pharmaceutical composition of claim 88 for use in the treatment of a primary tumor and / or for use in the prevention and / or treatment of metastasis.

90. 90. The multispecific antibody, nucleic acid construct(s), delivery vehicle or pharmaceutical composition for use according to any one of claims 88 to 89, wherein said cancer is a solid cancer such as a malignant solid tumor, for example an advanced and / or metastatic solid tumor.

91. 91. The multispecific antibody, nucleic acid construct(s), delivery vehicle or pharmaceutical composition for use according to any one of claims 88 to 90, wherein said cancer is carcinoma.

92. 91. The multispecific antibody, nucleic acid construct(s), delivery vehicle or pharmaceutical composition for use according to any one of claims 88 to 90, wherein said cancer expresses DR4.

93. 91. The multispecific antibody, one or more nucleic acid constructs, delivery vehicle or pharmaceutical composition for use according to any one of claims 88 to 90, wherein the tumor microenvironment comprises cancer-associated fibroblasts (CAFs).

94. 91. The multispecific antibody, one or more nucleic acid constructs, delivery vehicle or pharmaceutical composition for use according to any one of claims 86 to 90, wherein said CAFs express FAPα.

95. 94. The multispecific antibody, one or more nucleic acid constructs, delivery vehicle or pharmaceutical composition for use according to any one of claims 88 to 93, wherein said cancer expresses DR4 and the tumor microenvironment comprises CAFs, wherein said CAFs express FAPα.

96. 95. The multispecific antibody, nucleic acid construct(s), delivery vehicle or pharmaceutical composition for use according to any one of claims 88 to 94, wherein said cancer is selected from the group consisting of colorectal cancer [CRC], breast cancer, such as triple-negative breast cancer [TNBC], pancreatic cancer, such as pancreatic ductal adenocarcinoma [PDAC], esophagogastric cancer, such as gastric cancer and esophageal cancer, head and neck squamous cell carcinoma [HNSCC], cervical cancer, and lung cancer, such as non-small cell lung cancer [NSCLC].

97. 96. The multispecific antibody, one or more nucleic acid constructs, delivery vehicle or pharmaceutical composition for use according to any one of claims 88 to 95, wherein said cancer is selected from the group consisting of pancreatic cancer, gastric cancer and CRC.

98. 100. A method of treating cancer, comprising administering to a subject in need thereof an effective amount of a multispecific antibody according to any of claims 1 to 74, one or more nucleic acid constructs according to any of claims 75 to 77, a delivery vehicle according to any of claims 81 to 84, or a pharmaceutical composition according to claim 87.

99. 99. The method of claim 98, wherein the method is for treating a solid cancer, such as a malignant solid tumor, for example an advanced and / or metastatic solid tumor.

100. 100. The method of claim 99, wherein the cancer is a primary tumor and / or a metastasis.

101. 101. The method of any one of claims 99 to 100, wherein the cancer is selected from the group consisting of CRC, breast cancer, such as triple-negative breast cancer, pancreatic cancer, such as pancreatic ductal adenocarcinoma [PDAC], esophagogastric cancer, such as gastric cancer and esophageal cancer, head and neck squamous cell carcinoma [HNSCC], cervical cancer, and lung cancer, such as non-small cell lung cancer [NSCLC].

102. 102. The method of any one of claims 99 to 101, wherein the cancer is selected from the group consisting of pancreatic cancer, gastric cancer and colorectal cancer.

103. A method for producing an antibody as defined in any one of claims 1 to 74, comprising the steps of: (a) culturing the recombinant host cell of claims 84-85 under conditions in which the antibody is produced; and (b) isolating the antibodies produced from the culture; A method comprising:

104. A method for producing an antibody as defined in any one of claims 1 to 74, comprising the steps of: a) providing a first antibody comprising the FAPα-binding region of claim 1, and providing a second antibody comprising the DR4-binding region of claim 1; wherein said antibodies may contain the further features of claims 2-74, wherein said first and second antibodies comprise Fc regions, and wherein the sequences of the first and second CH3 regions of said first and second antibodies are different, whereby the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions; b) incubating the first antibody with the second antibody under reducing conditions sufficient to allow cysteines in the hinge region to undergo disulfide bond isomerization; and c) obtaining the antibody, wherein the antibody comprises a first immunoglobulin heavy chain and a first immunoglobulin light chain of the first antibody and a second immunoglobulin heavy chain and a second immunoglobulin light chain of the second antibody; The method includes:

105. (a)(i) a nucleic acid sequence encoding the heavy chain sequence of a FAPα binding region as defined in any one of claims 1 to 74; (ii) a nucleic acid sequence encoding the light chain sequence of the FAPα binding region defined in any one of claims 1 to 74 Culturing a host cell containing an expression vector comprising purifying the first antibody from the culture medium; (b)(iii) 75.) A nucleic acid sequence encoding the heavy chain sequence of a DR4 binding region as defined in any one of claims 1 to 74, (iv) a nucleic acid sequence encoding the light chain sequence of a DR4 binding region as defined in any one of claims 1 to 74 Culturing a host cell containing an expression vector comprising purifying the second antibody from the culture medium; (c) incubating the first antibody with the second antibody under reducing conditions sufficient to allow cysteines within the hinge region to undergo disulfide bond isomerization, thereby obtaining a bispecific antibody; 105. The method of claim 104, comprising:

106. 106. The method of claim 105, wherein step c) comprises adding a reducing agent.

107. 107. The method of claims 105-106, wherein step c) comprises the addition of a reducing agent selected from the group consisting of 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and beta-mercaptoethanol.

108. 108. The method of any one of claims 105 to 107, wherein step c) comprises the addition of a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine.

109. 109. A method according to any one of claims 105 to 108, wherein step c) comprises restoring conditions to non-reducing or less reducing, for example by removal of the reducing agent.

110. 110. The method of claim 109, wherein the reducing agent is removed by desalting.

111. A kit of parts, such as a kit for use as a companion diagnostic / for identifying patients within a patient population who have a propensity to respond to treatment with an antibody as defined in any one of claims 1 to 74, comprising an antibody as defined in any one of claims 1 to 74 and instructions for use of said kit.

112. A diagnostic composition comprising an antibody as defined in any one of claims 1 to 74.

113. 113. The diagnostic composition of claim 112, further comprising a dilution buffer.

114. An anti-FAPα antibody comprising at least one FAPα binding region, wherein the FAPα binding region comprises a heavy chain variable region (VH) comprising three complementarity determining regions CDR1, CDR2 and CDR3 present within the amino acid sequence shown in SEQ ID NO: 13, and a light chain variable region (VL) comprising three complementarity determining regions CDR1, CDR2 and CDR3 present within the amino acid sequence shown in SEQ ID NO:

14.

115. The anti-FAPα antibody of claim 114, wherein the anti-FAPα antibody comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 1, 2 and 3, respectively.

116. The anti-FAPα antibody of any one of claims 114 to 115, wherein the anti-FAPα antibody comprises a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 4, 5 and 6, respectively.

117. An anti-FAPα antibody according to any one of claims 114 to 116, wherein the anti-FAPα antibody comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 1, 2 and 3, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 4, 5 and 6, respectively.

118. An anti-FAPα antibody according to any one of claims 114 to 117, wherein the VH sequence of the FAPα binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO:

13.

119. An anti-FAPα antibody according to any one of claims 114 to 118, wherein the VL sequence of the FAPα binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO:

14.

120. An anti-FAPα antibody according to any one of claims 114 to 119, wherein the VH sequence of the FAPα binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 13, and the VL sequence of the FAPα binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:

14.

121. An anti-FAPα antibody according to any one of claims 114 to 120, wherein the VH sequence of the FAPα binding region differs from SEQ ID NO: 13 by up to 10 substitutions, such as up to 9 substitutions, for example up to 8 substitutions, such as up to 7 substitutions, for example up to 6 substitutions, such as up to 5 substitutions, for example up to 4 substitutions, such as up to 3 substitutions, for example up to 2 substitutions, such as up to 1 substitution.

122. An anti-FAPα antibody according to any one of claims 114 to 121, wherein the VL sequence of the FAPα binding region differs from SEQ ID NO: 14 by up to 10 substitutions, such as up to 9 substitutions, for example up to 8 substitutions, such as up to 7 substitutions, for example up to 6 substitutions, such as up to 5 substitutions, for example up to 4 substitutions, such as up to 3 substitutions, for example up to 2 substitutions, such as up to 1 substitution.

123. The anti-FAPα antibody of any one of claims 114 to 122, wherein the VH and VL sequences differ only in the framework regions.

124. The anti-FAPα antibody of any one of claims 114 to 123, wherein the antibody is monovalent.

125. An anti-FAPα antibody according to any one of claims 114 to 124, wherein the antibody is a bivalent antibody having two antigen-binding regions capable of binding to human FAPα, and wherein the two antigen-binding regions have the same variable region sequence.

126. (i) the anti-FAPα antibody comprises a first heavy chain and a second heavy chain; (ii) the first heavy chain and the second heavy chain each comprise at least a hinge region, a CH2 region, and a CH3 region; (iii) in the first heavy chain, at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (in a human IgG1 heavy chain according to Eu numbering) is substituted, and in the second heavy chain, at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (in a human IgG1 heavy chain according to Eu numbering) is substituted. An anti-FAPα antibody according to any one of claims 114 to 125.

127. The anti-FAPα antibody of any one of claims 114 to 126, wherein the first heavy chain and the second heavy chain are substituted at the same positions.

128. (i) the amino acid at the position corresponding to F405 (in the human IgG1 heavy chain according to Eu numbering) is L; or (ii) the amino acid at the position corresponding to K409 (in the human IgG1 heavy chain according to Eu numbering) is R; An anti-FAPα antibody according to any one of claims 114 to 127.

129. An anti-FAPα antibody according to any one of claims 114 to 128, wherein the anti-FAPα antibody comprises a first heavy chain and a second heavy chain, wherein one or both heavy chains are modified to reduce the extent of Fc-mediated effector function induced by the antibody compared to an identical antibody except for comprising unmodified first and second heavy chains.

130. The anti-FAPα antibody of any one of claims 114 to 129, comprising a first heavy chain and a second heavy chain, wherein in at least one of the first heavy chain and the second heavy chain, one or more amino acids at positions corresponding to positions L234, L235, G236, D265, N297, and P331 in a human IgG1 heavy chain according to Eu numbering are not L, L, G, D, N, and P, respectively.

131. An anti-FAPα antibody according to any one of claims 114 to 130, wherein the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to Eu numbering are F and E, respectively, in the first heavy chain and the second heavy chain.

132. An anti-FAPα antibody according to any one of claims 114 to 131, wherein the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain according to Eu numbering are F, E and A, respectively, in the first heavy chain and the second heavy chain.

133. An anti-FAPα antibody according to any one of claims 114 to 132, wherein the positions corresponding to positions L234, L235 and G236 in a human IgG1 heavy chain according to Eu numbering are F, E and R, respectively, in the first heavy chain and the second heavy chain.

134. the anti-FAPα antibody comprises a first heavy chain and a second heavy chain, wherein positions in both the first heavy chain and the second heavy chain corresponding to positions L234, L235, and G236 in a human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively; and wherein (i) the position corresponding to F405 in the human IgG1 heavy chain according to Eu numbering is L; or (ii) The anti-FAPα antibody according to any one of claims 114 to 133, wherein the position in the first heavy chain corresponding to K409 in a human IgG1 heavy chain according to Eu numbering is R.

135. The antibody (i) a FAPα binding region comprising a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 1, 2 and 3, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 4, 5 and 6, respectively; (ii) the anti-FAPα antibody comprises a first heavy chain and a second heavy chain, and wherein positions in both the first heavy chain and the second heavy chain corresponding to positions L234, L235, and G236 in a human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively; and (iii) the first heavy chain has an L at a position corresponding to F405 in a human IgG1 heavy chain according to Eu numbering, or an R at a position corresponding to K409 in a human IgG1 heavy chain according to Eu numbering, An anti-FAPα antibody according to any one of claims 114 to 134.

136. The antibody (i) a FAPα binding region comprising a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 1, 2 and 3, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 4, 5 and 6, respectively; (ii) the anti-FAPα antibody comprises a first heavy chain and a second heavy chain, and wherein positions in both the first heavy chain and the second heavy chain corresponding to positions L234, L235, and G236 in a human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively; and (iii) the position of the first heavy chain corresponding to F405 in a human IgG1 heavy chain according to Eu numbering is L; An anti-FAPα antibody according to any one of claims 114 to 134.

137. The anti-FAPα antibody of any one of claims 114 to 136, wherein the antibody comprises a heavy chain sequence shown in SEQ ID NO: 17 and a light chain sequence shown in SEQ ID NO:

18.

138. An anti-DR4 antibody comprising at least one DR4 binding region, the DR4 binding region comprising a heavy chain variable region (VH) comprising three complementarity determining regions CDR1, CDR2 and CDR3 present within the amino acid sequence set forth in SEQ ID NO: 15, and a light chain variable region (VL) comprising three complementarity determining regions CDR1, CDR2 and CDR3 present within the amino acid sequence set forth in SEQ ID NO:

16.

139. The anti-DR4 antibody of claim 138, wherein the DR4 binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 8 and 9, respectively.

140. The anti-DR4 antibody of any one of claims 138 to 139, wherein the DR4 binding region comprises a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, 11 and 12, respectively.

141. The anti-DR4 antibody of any one of claims 138 to 140, wherein the anti-DR4 binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 8 and 9, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, 11 and 12, respectively.

142. The anti-DR4 antibody of any one of claims 138 to 141, wherein the VH sequence of the DR4 binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:

15.

143. The anti-DR4 antibody of any one of claims 138 to 142, wherein the VL sequence of the DR4 binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:

16.

144. The anti-DR4 antibody of any one of claims 138 to 143, wherein the VH sequence of the DR4 binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 15, and the VL sequence of the DR4 binding region has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:

16.

145. The anti-DR4 antibody of any one of claims 138 to 144, wherein the VH sequence of the DR4 binding region differs from SEQ ID NO: 15 by up to 10 substitutions, such as up to 9 substitutions, for example up to 8 substitutions, such as up to 7 substitutions, for example up to 6 substitutions, such as up to 5 substitutions, for example up to 4 substitutions, such as up to 3 substitutions, for example up to 2 substitutions, such as up to 1 substitution.

146. The anti-DR4 antibody of any one of claims 138 to 145, wherein the VL sequence of the DR4 binding region differs from SEQ ID NO: 16 by up to 10 substitutions, such as up to 9 substitutions, for example up to 8 substitutions, such as up to 7 substitutions, for example up to 6 substitutions, such as up to 5 substitutions, for example up to 4 substitutions, such as up to 3 substitutions, for example up to 2 substitutions, such as up to 1 substitution.

147. The anti-DR4 antibody of any one of claims 138 to 146, wherein the VH and VL sequences differ only in the framework regions.

148. The anti-DR4 antibody of any one of claims 138 to 147, which is monovalent.

149. The anti-DR4 antibody of any one of claims 138 to 148, which is a bivalent antibody having two antigen-binding regions capable of binding to human DR4, and wherein the two antigen-binding regions have identical variable region sequences.

150. (i) the anti-DR4 antibody comprises a first heavy chain and a second heavy chain; (ii) the first heavy chain and the second heavy chain each comprise at least a hinge region, a CH2 region, and a CH3 region; (iii) in the first heavy chain, at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (in a human IgG1 heavy chain according to Eu numbering) is substituted, and in the second heavy chain, at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (in a human IgG1 heavy chain according to Eu numbering) is substituted. The anti-DR4 antibody of any one of claims 138 to 149.

151. The anti-DR4 antibody of any one of claims 138-150, wherein the first heavy chain and the second heavy chain are substituted at the same positions.

152. (i) the amino acid at the position corresponding to F405 (in the human IgG1 heavy chain according to Eu numbering) is L; or (ii) the amino acid at the position corresponding to K409 (in the human IgG1 heavy chain according to Eu numbering) is R; An anti-DR4 antibody according to any one of claims 138 to 151.

153. The anti-DR4 antibody of any one of claims 138 to 152, wherein the anti-DR4 antibody comprises a first heavy chain and a second heavy chain, wherein one or both heavy chains are modified to reduce the extent of Fc-mediated effector function that the antibody induces compared to an otherwise identical antibody comprising unmodified first and second heavy chains.

154. The anti-DR4 antibody of any one of claims 138 to 153, wherein the anti-DR4 antibody comprises a first heavy chain and a second heavy chain, wherein in at least one of the first heavy chain and the second heavy chain, one or more amino acids at positions corresponding to positions L234, L235, G236, D265, N297, and P331 in a human IgG1 heavy chain according to EU numbering are not L, L, G, D, N, and P, respectively.

155. The anti-DR4 antibody of any one of claims 138 to 154, wherein the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to Eu numbering are F and E in the first heavy chain and the second heavy chain, respectively.

156. The anti-DR4 antibody of any one of claims 138 to 155, wherein the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to Eu numbering are F, E, and A, respectively, in the first heavy chain and the second heavy chain.

157. The anti-DR4 antibody of any one of claims 138 to 156, wherein the positions corresponding to positions L234, L235, and G236 in a human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively, in the first heavy chain and the second heavy chain.

158. the anti-DR4 antibody comprises a first heavy chain and a second heavy chain, wherein positions in both the first heavy chain and the second heavy chain corresponding to positions L234, L235, and G236 in a human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively; and wherein (i) the position corresponding to F405 in the human IgG1 heavy chain according to Eu numbering is L; or (ii) the position of the first heavy chain corresponding to K409 in a human IgG1 heavy chain according to Eu numbering is R; The anti-DR4 antibody of any one of claims 138 to 157.

159. The antibody (i) a DR4 binding region comprising a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 8 and 9, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, 11 and 12, respectively; (ii) the anti-DR4 antibody comprises a first heavy chain and a second heavy chain, and the positions in both the first heavy chain and the second heavy chain that correspond to positions L234, L235, and G236 in a human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively; and (iii) in said first and second heavy chains, the position corresponding to K409 in a human IgG1 heavy chain according to Eu numbering is R, or in said first and second heavy chains, the position corresponding to F405 in a human IgG1 heavy chain according to Eu numbering is L; The anti-DR4 antibody of any one of claims 138 to 158.

160. The antibody (i) a DR4 binding region comprising a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 8 and 9, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, 11 and 12, respectively; (ii) the anti-DR4 antibody comprises a first heavy chain and a second heavy chain, and the positions in both the first heavy chain and the second heavy chain that correspond to positions L234, L235, and G236 in a human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively; and (iii) in the first and second heavy chains, the position corresponding to K409 in a human IgG1 heavy chain according to Eu numbering is R; The anti-DR4 antibody of any one of claims 138 to 158.

161. The anti-DR4 antibody of any one of claims 138 to 160, wherein the antibody comprises a heavy chain sequence shown in SEQ ID NO: 19 and a light chain sequence shown in SEQ ID NO:

20.

162. (a)(i) a first antibody according to any one of claims 114 to 137, and A second antibody comprising a DR4 binding region comprising a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 8 and 9, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, 11 and 12, respectively; a first and a second antibody, wherein the antibodies comprise a first heavy chain and a second heavy chain, and the positions in both the first heavy chain and the second heavy chain that correspond to positions L234, L235, and G236 in a human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively; (ii) a second antibody according to any one of claims 138 to 161, and A first antibody comprising a FAPα binding region comprising a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 1, 2 and 3, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 4, 5 and 6, respectively; a first and a second antibody, wherein the antibodies comprise a first heavy chain and a second heavy chain, and wherein positions in both the first heavy chain and the second heavy chain corresponding to positions L234, L235, and G236 in a human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively; or (iii) a first antibody according to any one of claims 114 to 137 and a second antibody according to any one of claims 138 to 161; providing wherein the sequences of the first and second CH3 regions of said first and second antibodies are different, such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions, and preferably, the amino acid at the position corresponding to F405 in said first CH3 region is L and the amino acid at the position corresponding to K409 in said second CH3 region is R, or vice versa; (b) incubating the first antibody with the second antibody under reducing conditions sufficient to allow cysteines in the hinge region to undergo disulfide bond isomerization; and (c) obtaining a multispecific antibody comprising the first immunoglobulin heavy chain and the first immunoglobulin light chain of said first antibody and the second immunoglobulin heavy chain and the second immunoglobulin light chain of said second antibody; A method for producing a multispecific antibody, comprising:

163. (a)(i) a first antibody according to any one of claims 114 to 137, and a second antibody comprising a DR4-binding region, wherein the heavy chain variable region (VH) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 8 and 9, respectively, and the light chain variable region (VL) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, 11 and 12, respectively; a first and a second antibody, wherein the antibodies comprise a first heavy chain and a second heavy chain, and the positions in both the first heavy chain and the second heavy chain that correspond to positions L234, L235, and G236 in a human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively; (ii) a second antibody according to any one of claims 138 to 161, and A first antibody comprising a FAPα-binding region, wherein the heavy chain variable region (VH) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 1, 2 and 3, respectively, and the light chain variable region (VL) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 4, 5 and 6, respectively; a first and a second antibody, wherein the antibodies comprise a first heavy chain and a second heavy chain, and wherein positions in both the first heavy chain and the second heavy chain corresponding to positions L234, L235, and G236 in a human IgG1 heavy chain according to Eu numbering are F, E, and R, respectively; or (iii) a first antibody according to any one of claims 114 to 137 and a second antibody according to any one of claims 138 to 161; providing wherein the sequences of the first and second CH3 regions of the first and second antibodies are different, such that the heterodimeric interaction between the first and second CH3 regions is stronger than each of the homodimeric interactions of the first and second CH3 regions, and preferably the amino acid at the position corresponding to F405 in the first CH3 region is L and the amino acid at the position corresponding to K409 in the second CH3 region is R; (b) incubating the first antibody with the second antibody under reducing conditions sufficient to allow cysteines in the hinge region to undergo disulfide bond isomerization; and (c) obtaining a multispecific antibody comprising the first immunoglobulin heavy chain and the first immunoglobulin light chain of the first antibody, and the second immunoglobulin heavy chain and the second immunoglobulin light chain of the second antibody; 163. A method for producing a multispecific antibody according to claim 162, comprising:

164. A multispecific antibody obtainable by the method of any one of claims 162 to 163.

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